Method for preventing, improving, or treating inflammatory bowel disease

ABSTRACT

Pyrrolidine carboxamido derivatives, optical isomers thereof, and salts thereof that are able to prevent, improve, and/or treat inflammatory conditions, including inflammatory bowel disease, and methods for preparing and using the same are provided.

CROSS REFERENCE TO RELATED APPLICATION

This application is a divisional application of U.S. application Ser.No. 15/205,853 filed on Jul. 08, 2016, which claims priority to KoreanApplication No. 10-2015-0097040 filed on Jul. 8, 2015. The applicationsare incorporated herein by reference.

INCORPORATION-BY-REFERENCE OF SEQUENCE LISTING

The contents of the sequence listing text file named “050572-501D01US_ST25.txt”, which was created on Mar. 13, 2018 and is 5,619 bytes insize, is hereby incorporated by reference in its entirety.

TECHNICAL FIELD

The present invention relates to pyrrolidine carboxamido derivatives,optical isomers thereof, or pharmaceutically acceptable salts thereof,and methods for preparing and using the same.

BACKGROUND ART

Various compounds/compositions/methods including, but not limited to,immunosuppressive drugs (e.g., infliximab), aminosalicylic acids (e.g.,sulfasalazine), and steroids have been proposed as means for reducingcytokines and/or chemokines to prevent and/or treat various diseasesincluding, but not limited to, inflammatory indications, cancers, andophthalmic indications (Expert opinion on emerging drugs (2015)20(3):349-352; Cell. (2010) Mar. 19; 140(6): 883-899; Progress inRetinal and Eye Research 37(2013) 68e89, which are incorporated hereinby reference). They are, however, unsatisfactory at least because theyare expensive, and/or involve side effects, and/or show low therapeuticefficacy (P&T 41(2016), Jun. no 6; Gut 56(2007):725-732; World JGastroenterol (2005);11(16):2462-2466, which are incorporated herein byreference). Therefore, there remains a need for a new compound,composition, and/or a method.

SUMMARY

The present invention is based on the discovery that certain pyrrolidinecarboxamido derivatives are able to suppress the expression and activityof inflammatory cytokines (e.g., IL-6) and/or chemokines and are able toremain at a sufficiently high concentration in a target tissue/cellwhile being less exposed to blood. The present invention is also basedon the discovery that certain pyrrolidine carboxamido derivatives areable to inhibit the activity of NF-κB by stabilizing of IκB. The presentinvention is further based on the discovery that certain pyrrolidinecarboxamido derivatives are able to disrupt the formation ofinflammatory signal transduction complex mediated by myeloiddifferentiation primary response gene 88 (MyD88) and/orreceptor-interacting protein 1 (RIP1) that act in the downstream ofsignaling pathway involving toll-like receptor 2/4 and IL-1β.

In one aspect, the present invention provides compounds represented bythe following Formula 1, optical isomers thereof, or pharmaceuticallyacceptable salts thereof.

wherein: n is 0, 1, or 2; A is -a¹-, which is an amino acidindependently selected from the group consisting of alanine, (Ala, A),arginine (Arg, R), asparagine (Asn, N), aspartic acid (Asp, D), cysteine(Cys, C), glutamic acid (Glu, E), glutamine (Gln, Q), glycine (Gly, G),histidine (His, H), isoleucine (Ile, I), leucine (Leu, L), lysine (Lys,K), methionine (Met, M), phenylalanine (Phe, F), proline (Pro, P),serine (Ser, S), threonine (Thr, T), tryptophan (Trp, W), tyrosine (Tyr,Y), and valine (Val, V), both terminal ends of the amino acid beingcoupled to a carbonyl group or an amine group by an amide bond; and R¹is a straight chain or branched chain C₁₋₃₆ alkyl, a straight chain orbranched chain C₂₋₃₆ alkenyl including at least one double bond, or astraight chain or branched chain C₂₋₃₆ alkynyl including at least onetriple bond.

In another aspect, the present invention provides methods for preparingthe compounds, the optical isomers, and the salts.

In still another aspect, the present invention provides compositions forpreventing, improving, and treating various diseases (e.g., inflammatoryindications, cancers, and ophthalmic indications). The compositions eachcomprise, as an active component, at least one of the compounds, atleast one of the optical isomers, or at least one of the salts.

In still yet another aspect, the present invention provides methods forpreventing, improving, or treating various diseases (e.g., inflammatoryindications, cancers, and ophthalmic indications). The methods eachcomprise administering to a subject in need a composition containing, asan active component, at least one of the compounds, at least one of theoptical isomers, or at least one of the salts.

Compounds according to certain embodiments of the present invention mayinhibit decomposition of IκB in inflammation signaling pathway mediatedby MyD88 (myddosome complex) and/or RIP 1, thereby preventing NF-κB frombeing transported into nucleus of a cell, resulting in suppression ofexpression of cytokines and chemokines (e.g., G-CSF, IL-2, SCF, VEGF,CX3CL1, IGFBP5, IGFBP6, IL-1α, IL-1β, IL-6, IL-9, MCP-1, MIP-3α,IL12p40/70, MIG, TNF-α, and VCAM-1) and preventing inflammation reactionthat could otherwise be caused by the expression thereof.

Other aspects and advantages of the present invention will becomeapparent to the skilled in the art from a consideration of the detaileddescription and the drawings.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1A is an electrophoresis result showing that compounds according toembodiments of the present invention suppress the expression of IL-6.(Compound 1.1 (SEQ ID NO: 2)).

FIG. 1B is a graph showing that compounds according to embodiments ofthe present invention suppress the expression of IL-6. (Compound 1.1(SEQ ID NO: 2)).

FIGS. 2A-2D are bar graphs that show that compounds according toembodiments of the present invention suppress the expression ofcytokines and chemokines in a cell line RAW 264.7. FIG. 2A is a bargraph that shows that the expression of GCSF, IL-2, SCF, and VEGF wassuppressed as statistically meaningful when the cells were treated withthe compounds of the present invention. FIG. 2B is a bar graph thatshows that the expression of CX3CL1, IGFBP5, IGFBP6, IL-1 b, IL-6, andIL-9 was suppressed as statistically meaningful when the cells weretreated with the compounds of the present invention. FIG. 2C is a bargraph that shows that the expression of MCP-1, MIP-3a, IL12p40/70, MIG,TNF-a, and VCAM-1 was suppressed as statistically meaningful when thecells were treated with the compounds of the present invention. FIG. 2Dis a bar graph that shows that the expression of IL-1a was suppressed asstatistically meaningful when the cells were treated with the compoundsof the present invention.

FIG. 3 shows that compounds according to embodiments of the presentinvention suppress the expression of IL-6 in a host in a cell line RAW264.7. (Compound 1.1 (SEQ ID NO: 2)).

FIG. 4 shows that compounds according to embodiments of the presentinvention inhibit the activity of NF-κB. (Compound 1.1 (SEQ ID NO: 2)).

FIG. 5 shows that the compounds according to embodiments of the presentinvention suppress NF-κB. (Compound 1.1 (SEQ ID NO: 2), Referencecompound 1 (SEQ ID NO: 18)).

FIGS. 6A-6C are bar graphs that show that the compounds according toembodiments of the present invention inhibit the activity of NF-κB (FIG.6A) while not affecting signal transmission of TGF-β (FIG. 6B) and BMP(FIG. 6C). (Compound 1.1 (SEQ ID NO: 2)).

FIG. 7 is an immunoprecipitation result showing that the compoundsaccording to embodiments of the present invention inhibit formation ofinflammation signaling pathway protein complex mediated by IRAK-1,MyD88, and/or RIP1 and showing that the compounds according toembodiments of the present invention change the concentration of IκB.(Compound 1.1 (SEQ ID NO: 2)).

FIG. 8 is an immunoprecipitation result showing that the compoundsaccording to embodiments of the present invention can disrupt formationof inflammation signaling pathway protein complex mediated by IRAK-1,MyD88, and/or RIP1. (Compound 1.1 (SEQ ID NO: 2), Reference compound 1(SEQ ID NO: 18)).

FIGS. 9A and 9B show that change in pretreatment concentration of thecompounds according to embodiments of the present invention changeconcentration of IκB in RAW 264.7 macrophage cells (FIG. 9A) and BMDMcells (FIG. 9B). (Compound 1.1 (SEQ ID NO: 2), Reference compound 1 (SEQID NO: 18)).

FIG. 10 is a graph indicating disease activity index scores in an animalmodel with DSS-induced chronic colitis according to the dose ofcompounds according to embodiments of the present invention in case oforal administration thereof. (Compound 1.1 (SEQ ID NO: 2)).

FIG. 11A shows disease activity index scores representing the ability ofcompounds according to embodiments of the present invention to suppressacute colitis in an animal model with DSS-induced acute colitis.(Compound 1.1 (SEQ ID NO: 2)).

FIGS. 11B-11D shows the compounds according to embodiments of thepresent invention affect the amount of expression of chemokines (CCL2(FIG. 11C), CCL20 (FIG. 11B), and CXCL1 (FIG. 11D)) in a mice model withDSS-induced chronic colitis. (Compound 1.1 (SEQ ID NO: 2)).

FIG. 12 are images showing shapes of large intestinal villi from anon-treated group.

FIG. 13 are images showing shapes of large intestinal villi from aDDS-induced chronic colitis model group.

FIG. 14 are images showing shapes of large intestine villi from a grouptreated with a compound according to embodiments of the presentinvention. (Compound 1.1 (SEQ ID NO: 2))

FIG. 15 are images of large intestinal tissues obtained from non-treatedgroup, the DDS-induced chronic colitis model group, the group treatedwith compound 1.1 (SEQ ID NO: 2) (mpk) and the group treated withsulfasalazine (500 mpk) as an anti-inflammatory drug for colitistreatment.

FIG. 16 are images showing the morphology of large intestinal mucousmembranes obtained from the non-treated group, the DDS-induced chroniccolitis model group, the treated group with compound 1.1 (SEQ ID NO: 2)(100 mpk) and the group treated with sulfasalazine (500 mpk) as ananti-inflammatory drug for colitis treatment.

FIG. 17 is a graph showing recovery level of large intestinal wall in anon-treated group, a DDS-induced chronic colitis model group, a grouptreated with compounds according to embodiments of the present invention(Compound 1.1 (SEQ ID NO: 2)), and a group treated with sulfasalazine.

FIG. 18 is a graph showing changes in blood concentration over time ofcompounds according to embodiments of the present invention (Compound1.1 (SEQ ID NO: 2)) via intravenous administration.

FIG. 19 is a graph showing changes in blood concentration over time ofcompounds according to embodiments of the present invention (Compound1.1 (SEQ ID NO: 2)) via oral administration.

FIG. 20A is a Western blot image confirming whether compounds accordingto embodiments of the present invention (Compound 1.1 (SEQ ID NO: 2))inhibit MAPK/ERK signaling pathway.

FIG. 20B is a diagram depicting signaling pathway of toll-likereceptors.

FIG. 21 is a Western blot image confirming whether compounds accordingto embodiments of the present invention inhibit MAPK/ERK signalingpathway. (Compound 1.1 (SEQ ID NO: 2), Reference compound 1 (SEQ ID NO:18)).

FIG. 22A and FIG. 22B show the inhibition level of NF-κB activation bycompounds according to embodiments of the present invention (FIG. 22A)(Compound 1.1 (SEQ ID NO: 2)) and an IRAK1/4 inhibitor (FIG. 22B),respectively.

FIGS. 23A and 23B are immunoblot images confirming whether compoundsaccording to embodiments of the present invention and an IRK1/4inhibitor change the concentration of IκB. FIG. 23A is an immunoblotimage showing cells pretreated with compound 1.1 (SEQ ID NO: 2) (100nM), IRAK1/4 inhibitor (25 μM) and smaducin-6 (100 nM) and then furthertreated with LPS (100 ng/m1). FIG. 23B is an immunoblot image showingthat RAK1/4 inhibitor also suppressed degradation of IκB similar tocompound 1.1 (SEQ ID NO: 2).

FIG. 24A and FIG. 24B are an image and a graph, respectively, comparingthe ability of compounds according to embodiments of the presentinvention to inhibit MAPK/ERK signaling pathway and the ability of anIRAK1/4 inhibitor to inhibit MAPK/ERK signaling pathway. (Compound 1.1(SEQ ID NO: 2))

FIGS. 25A and 25B are Western blot images confirming whether compoundsaccording to embodiments of the present invention suppress, in ARPE-19,expression of Nox-4, VEGF, VEGFR1, VEGFR2, Ang-2, EPO, and EPOR and canincrease the expression of Ang-1 and Tie2. FIG. 25A is a Western blotimage confirming that compounds according to embodiments of the presentinvention (Compound 1.1 (SEQ ID NO: 2)) suppress, in ARPE-19, expressionof Nox-4, VEGF, VEGFR1, and VEGFR2. FIG. 25B is a Western blot imageconfirming that compounds according to embodiments of the presentinvention (Compound 1.1 (SEQ ID NO: 2)) suppress, in ARPE-19, Ang-2,EPO, and EPOR, and can increase expression of Ang-1 and Tie2.

FIG. 25C is a qRT-PCR image confirming whether compounds according toembodiments of the present invention (Compound 1.1 (SEQ ID NO: 2))suppress the expression of VEGF in HRMEC.

FIG. 26 is an image showing that compounds according to embodiments ofthe present invention (Compound 1.1 (SEQ ID NO: 2)) suppress tubeformation in HRMEC.

FIGS. 27A and 27B are images showing that compounds according toembodiments of the present invention suppress activated oxygen increasedin a mice model with STZ-induced type 1 diabetic retinopathy. FIG. 27Ashows an image of the administration schedule. FIG. 27B are imagesshowing retina tissues stained with 5 μM of dihydroethidium and measuredactive oxygen rates from each group. (Compound 1.1 (SEQ ID NO: 2)).

FIG. 28A is a graph showing that compounds according to embodiments ofthe present invention have a therapeutic effect in MOG-induced EAE mice.(Compound 1.1 (SEQ ID NO: 2)).

FIG. 28B is a graph showing that compounds according to embodiments ofthe present invention change the weight of MOG-induced EAE mice.(Compound 1.1 (SEQ ID NO: 2)).

FIG. 29 is a graph showing that compounds according to embodiments ofthe present invention have a therapeutic effect in a Cecal ligation andpuncture (CLP) model. (Compound 1.1 (SEQ ID NO: 2)).

DETAILED DESCRIPTION

1. Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the meaning commonly understood by a person skilled in the art towhich this disclosure belongs. The following references, which areincorporated herein by reference, provide one of skill with a generaldefinition of many of the terms used in this invention: The CambridgeDictionary of Science and Technology (Walker ed., 1988); The Glossary ofGenetics, 5th Ed., R. Rieger et. al. (eds.), Springer Verlag (1991); andHale & Marham, The Harper Collins Dictionary of Biology (1991). As usedherein, the following terms have the meanings ascribed to them below,unless specified otherwise.

Unless specifically stated or obvious from context, as used herein, theterm “or” is understood to be inclusive.

Unless specifically stated or obvious from context, as used herein, theterms “a”, “an”, and “the” are understood to be singular or plural.Thus, for example, reference to “a compound” includes mixtures of suchcompounds; reference to “a carrier” includes mixtures of two or morecarriers; and the like.

Unless specifically stated or obvious from context, as used herein, theterm “about” is understood as within a range of normal tolerance in theart, for example within 2 standard deviations of the mean. About can beunderstood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%,0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear fromcontext, all numerical values provided herein are modified by the termabout.

The terms “active agent,” “drug,” and “pharmaceutical agent” are usedinterchangeably herein to refer to a chemical material or compoundwhich, when administered to a subject (e.g., any animal including ahuman or non-human animal) by any means and/or routes induces a desiredpharmacologic effect (e.g., such as a reduction of inflammation).

The term “additive” as used herein may refer to any additionalcomponents that may be added to the compositions described herein. Forexample, additives may include excipients (e.g., one or moreexcipients), antioxidants (e.g., one or more antioxidants), stabilizers(e.g., one or more stabilizers), preservatives (e.g., one or morepreservatives), pH adjusting and/or buffering agents (e.g., one or morepH adjusting and/or buffering agents), tonicity adjusting agents (e.g.,one or more tonicity adjusting agents), thickening agents (e.g., one ormore thickening agents), suspending agents (e.g., one or more suspendingagents), binding agents (e.g., one or more binding agents),viscosity-increasing agents (e.g., one or more viscosity-increasingagents), and the like, provided that the additional components arepharmaceutically acceptable for the particular condition to be treated.The additives may also include processing agents and drug deliverymodifiers and enhancers, such as, for example, calcium phosphate,magnesium stearate, talc, monosaccharides, disaccharides, starch,gelatin, cellulose, methyl cellulose, sodium carboxymethyl cellulose,dextrose, hydroxypropyl-beta-cyclodextrin, polyvinylpyrrolidinone, lowmelting waxes, ion exchange resins, and the like, as well ascombinations of any two or more thereof. Other suitable pharmaceuticallyacceptable excipients are described in “Remington's PharmaceuticalSciences,” Mack Pub. Co., New Jersey (1991), and “Remington: The Scienceand Practice of Pharmacy,” Lippincott Williams & Wilkins, Philadelphia,20th edition (2003) and 21st edition (2005), which are incorporatedherein by reference. The additives described herein may be used in anysuitable amounts.

As used herein, the term “administering” means oral administration,administration as a suppository, topical contact, intravenous,parenteral, intraperitoneal, intramuscular, intralesional, intrathecal,intranasal or subcutaneous administration, or the implantation of aslow-release device, e.g., a mini-osmotic pump, to a subject.Administration is by any route, including parenteral and transmucosal(e.g., oral, nasal, pulmonary, rectal, buccal, vaginal, ocular andtransdermal routes).

The terms “derivative” and ‘analog” are used herein interchangeably, andrefer to a compound that possesses the same core as a parent compound,but differs from the parent compound in bond order, in the absence orpresence of one or more atoms and/or groups of atoms, and combinationsthereof. The derivative can differ from the parent compound, forexample, in one or more substituents present on the core, which mayinclude one or more atoms, functional groups, or substructures. Thederivative can also differ from the parent compound in the bond orderbetween atoms within the core. In general, a derivative can be imaginedto be formed, at least theoretically, from the parent compound viachemical and/or physical processes.

As used herein, “antioxidants” may refer to are man-made or naturalsubstances that may prevent or delay some types of cell damage and/oroxidation. Antioxidants are found in many foods, including fruits andvegetables. They are also available as dietary supplements. Exemplaryantioxidants may include: βeta-carotene, Lutein, Lycopene, Selenium,Vitamin A, Vitamin C, and Vitamin E. Other antioxidants known to one ofskill in the art may also be used. The antioxidants described herein maybe used in any suitable amount.

By “co-administer” it is meant that a compound or composition describedherein is administered at the same time, just prior to, or just afterthe administration of additional therapies or active agents or additivesdescribed herein. The compound or the composition of the disclosure canbe administered alone or can be co-administered to a subject in need.Co-administration is meant to include simultaneous or sequentialadministration of the compound individually or in combination (more thanone compound or agent). The preparations can also be combined, whendesired, with other active substances.

In this disclosure, “comprises,” “comprising,” “containing” and “having”and the like can have the meaning ascribed to them in U.S. Patent lawand can mean “ includes,” “including,” and the like; “consistingessentially of” or “consists essentially” likewise has the meaningascribed in U.S. Patent law and the term is open-ended, allowing for thepresence of more than that which is recited so long as basic or novelcharacteristics of that which is recited is not changed by the presenceof more than that which is recited, but excludes prior art embodiments.

As used herein, “concurrent administration” includes overlapping induration at least in part. For example, when two agents (e.g., any ofthe agents or class of agents described herein that has bioactivity) areadministered concurrently, their administration occurs within a certaindesired time. The agents' administration may begin and end on the sameday. The administration of one agent can also precede the administrationof a second agent by day(s) as long as both agents are taken on the sameday at least once. Similarly, the administration of one agent can extendbeyond the administration of a second agent as long as both agents aretaken on the same day at least once. The active agent(s) does not haveto be taken at the same time each day to include concurrentadministration.

As used herein, an “effective amount” or “therapeutically effectiveamount” is that amount sufficient to affect a desired biological effect,such as beneficial results, including clinical results. As such, an“effective amount” depends upon the context in which it is beingapplied. An effective amount may vary according to factors known in theart, such as the disease state, age, sex, and weight of the individualbeing treated. Several divided doses may be administered daily or thedose may be proportionally reduced as indicated by the exigencies of thetherapeutic situation. In addition, the compounds, compositions, orformulations of this disclosure can be administered as frequently asnecessary to achieve a therapeutic amount.

The term, “gel” as used herein may refer to a material which is not areadily flowable liquid and not a solid, i.e., semi-solid. Gels may beformed from naturally occurring or synthetic materials. The gels can benon-ordered to slightly ordered showing some birefringence, liquidcrystal character. Gels may be administered topically.

The term “inflammatory bowel disease” as used herein has its usualmedical meaning, and refers to a group of inflammatoryindications/conditions of a colon and small intestine. Exemplaryinflammatory bowel diseases may include, but are not limited to, Crohn'sdisease, ulcerative colitis, Johne's disease, Behget's syndrome,collagenous colitis, diversion colitis, indeterminate colitis, infectivecolitis, ischaemic colitis, lymphocytic colitis, and closely relateddiseases and disorders of the gastrointestinal tract.

The term “inhibit,” as used herein, means to prevent, decrease,slow-down or arrest. In one embodiment, a compound, composition, orformulation may be considered to inhibit the viability of at least oneprotein (e.g., G-CSF, IL-2, SCF, VEGF, CX3CL1, IGFBP5, IGFBP6, IL-1 α,IL-1β, IL-6, IL-9, MCP-1, MIP-3α, IL12p40/70, MIG, TNF-α, VCAM-1, andNF-κB) when the amount or rate of the process or reaction that takesplace in the presence of the compound, composition, or formulation isdecreased by at least about 10% when compared to the amount or rate inthe absence of the compound, composition, or formulation. In anotherembodiment, a compound, composition, or formulation may be considered toinhibit a process or reaction when the amount or rate of the process orreaction that takes place in the presence of the compound, composition,or formulation is decreased by at least about 20% when compared to theamount or rate in the absence of the compound, composition, orformulation. In other embodiments, a compound, composition, orformulation may be considered to inhibit viability of one or moreproteins (e.g., G-CSF, IL-2, SCF, VEGF, CX3CL1, IGFBP5, IGFBP6, IL-1α,IL-1β, IL-6, IL-9, MCP-1, MIP-3α, IL12p40/70, MIG, TNF-α, VCAM-1, andNF-κB) when the amount or rate of viability that takes place in thepresence of the compound, composition, or formulation is decreased by atleast about 25%, about 30%, about 40%, about 50%, about 60%, about 70%,about 75% or about 80% when compared to the amount or rate in theabsence of the compound, composition, or formulation. In still otherembodiments, a compound, composition, or formulation may be consideredto inhibit viability of one or more proteins, i.e. arresting itsdevelopment.

As used herein, “intermittent administration” includes theadministration of an active agent for a period of time (which can beconsidered a “first period of administration”), followed by a timeduring which the agent is not taken or is taken at a lower maintenancedose (which can be considered “off-period”) followed by a period duringwhich the agent is administered again (which can be considered a “secondperiod of administration”). Generally, during the second phase ofadministration, the dosage level of the agent will match thatadministered during the first period of administration but can beincreased or decreased as medically necessary.

“Jelly” according to the current disclosure is a class of gels, whichare semisolid systems that consist of suspensions made up either smallinorganic particles or large organic molecules interpenetrated by aliquid, in which the structural coherent matrix contains a high portionof liquid, usually water.

“Liquid” as used herein is a dosage form consisting of a composition inits liquid state. A liquid is pourable; it flows and conforms to itscontainer at room temperature. Liquids display Newtonian orpseudoplastic flow behavior. In embodiments, a “semi-liquid” as usedherein may have properties of both a liquid and another formulation(i.e., a suspension, an emulsion, a solution, a cream, a gel, a jelly,and the like).

“Myeloid differentiation primary response gene 88” or “MYD88” is aprotein that, in humans, is encoded by the MYD88 gene. MyD88 plays acentral role in the innate and adaptive immune response. This proteinfunctions as an essential signal transducer in the interleukin-1 andToll-like receptor signaling pathways. These pathways regulate thatactivation of numerous proinflammatory genes. The encoded proteinconsists of an N-terminal death domain and a C-terminalToll-interleukin1 receptor domain.

As used herein, the term “ointment” may refer to a highly viscous liquidor semi-liquid formulation that may be used for therapeutic treatment ofa disease, syndrome, or condition (e.g., inflammatory bowel disease).

As used herein “pharmaceutically acceptable carrier” includes any andall solvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents, and the like that arephysiologically compatible. The type of carrier can be selected basedupon the intended route of administration. Pharmaceutically acceptablecarriers include sterile aqueous solutions or dispersions and sterilepowders for the extemporaneous preparation of sterile topical solutionsor dispersion. The use of such media and agents for pharmaceuticallyactive substances is well known in the art. Except insofar as anyconventional media or agent is incompatible with the composition (e.g.,Formula I (SEQ ID NO: 1) as described herein, derivatives/analogues ofFormula I (SEQ ID NO: 1), or a pharmaceutically acceptable salt,solvent, hydrate, or polymorph thereof), use thereof in the ophthalmiccompositions for the disclosure is contemplated.

“Pharmaceutical carriers” or “carriers” as used herein can furtherinclude pharmaceutically acceptable carriers, excipients, or stabilizerswhich are nontoxic to the cell or mammal being exposed thereto at thedosages and concentrations employed. Often the physiologicallyacceptable carrier is an aqueous pH buffered solution. Examples ofphysiologically acceptable carriers include buffers such as phosphate,citrate, and other organic acids; antioxidants including ascorbic acid;low molecular weight (less than about 10 residues) polypeptide;proteins, such as serum albumin, gelatin, or immunoglobulins;hydrophilic polymers such as polyvinylpyrrolidone; amino acids such asglycine, glutamine, asparagine, arginine or lysine; monosaccharides,disaccharides, and other carbohydrates including glucose, mannose, ordextrins; chelating agents such as EDTA; sugar alcohols such as mannitolor sorbitol; salt-forming counterions such as sodium; and/or nonionicsurfactants such as Tween™, polyethylene glycol (PEG), and Pluronics™.

Additionally, “pharmaceutically acceptable” means approved or approvableby a regulatory agency of the Federal or a state government or thecorresponding agency in countries other than the United States, or thatis listed in the U.S. Pharmacopoeia or other generally recognizedpharmacopoeia for use in animals, and more particularly, in humans.

The terms, “pH agent” or “buffering agent” as used herein may refer tocompounds or buffers useful as pH regulators. These include, but are notlimited to, glycerol buffers, citrate buffers, borate buffers, acetatebuffers, gluconate buffers, phosphate buffers, or citric acid-phosphatebuffers may also be included. The pH agent or buffering agent may beused in any suitable amount.

The term, “preservative” as described herein may refer to a substance orchemical that prevents undesirable chemical changes of the compound orcompositions or formulas described herein. Suitable preservatives mayinclude, for example, benzalkonium chloride, thimerosal, chlorobutanol,methyl paraben, propyl paraben, phenylethyl alcohol, edetate disodiumsorbic acid, Onamer M Polyquat, cetyl bromide, cetyl pyridiniumchloride, benzyl bromide, EDTA, phenylmercury nitrate, phenylmercuryacetate, thimerosal, merthiolate, acetate and phenylmercury borate,polymyxin B sulphate, methyl and propyl parabens, quaternary ammoniumchloride, sodium benzoate, sodium proprionate, and sodium perborate, andother agents known to those skilled in the art, or a combinationthereof. The preservative may be used in any suitable amount.

The terms “prevent,” “preventing,” or “prevention,” and othergrammatical equivalents as used herein, include to keep from developing,occur, hinder or avert a disease or condition symptoms as well as todecrease the occurrence of symptoms. The prevention may be complete(i.e., no detectable symptoms) or partial, so that fewer symptoms areobserved than would likely occur absent treatment. The terms furtherinclude a prophylactic benefit. For a disease or condition to beprevented, the compositions may be administered to a patient at risk ofdeveloping a particular disease, or to a patient reporting one or moreof the physiological symptoms of a disease, even though a diagnosis ofthis disease may not have been made.

Ranges provided herein are understood to be shorthand for all of thevalues within the range. For example, a range of 1 to 50 is understoodto include any number, combination of numbers, or sub-range from thegroup consisting 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 aswell as all intervening decimal values between the aforementionedintegers such as, for example, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8,and 1.9. With respect to sub-ranges, “nested sub-ranges” that extendfrom either end point of the range are specifically contemplated. Forexample, a nested sub-range of an exemplary range of 1 to 50 maycomprise 1 to 10, 1 to 20, 1 to 30, and 1 to 40 in one direction, or 50to 40, 50 to 30, 50 to 20, and 50 to 10 in the other direction. Rangescan be expressed herein as from “about” one particular value, and/or to“about” another particular value. When such a range is expressed,another aspect includes from the one particular value and/or to theother particular value. Similarly, when values are expressed asapproximations, by use of the antecedent “about,” it is understood thatthe particular value forms another aspect. It is further understood thatthe endpoints of each of the ranges are significant both in relation tothe other endpoint, and independently of the other endpoint. It is alsounderstood that there are a number of values disclosed herein, and thateach value is also herein disclosed as “about” that particular value inaddition to the value itself. It is also understood that throughout theapplication, data are provided in a number of different formats and thatthis data represent endpoints and starting points and ranges for anycombination of the data points. For example, if a particular data point“10” and a particular data point “15” are disclosed, it is understoodthat greater than, greater than or equal to, less than, less than orequal to, and equal to 10 and 15 are considered disclosed as well asbetween 10 and 15. It is also understood that each unit between twoparticular units are also disclosed. For example, if 10 and 15 aredisclosed, then 11, 12, 13, and 14 are also disclosed.

“Receptor interacting protein” or “RIP1” as used herein describes aprotein kinase which is a crucial regulator of cell survival and death.RIP1 and RIP2 also bear a C-terminal domain belonging to the deathdomain superfamily, allowing recruitment to large protein complexesinitiating different signaling pathways.

As used herein, “salts” or “salt form” or “pharmaceutically acceptedsalts” may include base addition salts (formed with free carboxyl orother anionic groups) which are derived from inorganic bases such as,for example, sodium, potassium, ammonium, calcium, or ferric hydroxides,and such organic bases as isopropylamine, triethylamine,2-ethylamino-ethanol, histidine, procaine, and the like. Such salts areformed as acid addition salts with any free cationic groups andgenerally are formed with inorganic acids such as, for example,hydrochloric, sulfuric, or phosphoric acids, or organic acids such asacetic, citric, p-toluenesulfonic, methanesulfonic acid, oxalic,tartaric, mandelic, and the like. Salts of the disclosure may includeamine salts formed by the protonation of an amino group with inorganicacids such as hydrochloric acid, hydrobromic acid, hydroiodic acid,sulfuric acid, phosphoric acid, and the like. Salts of the disclosurealso include amine salts formed by the protonation of an amino groupwith suitable organic acids, such as p-toluenesulfonic acid, aceticacid, and the like. Additional excipients which are contemplated for usein the practice of the present disclosure are those available to thoseof ordinary skill in the art, for example, those found in the UnitedStates Pharmacopoeia Vol. XXII and National Formulary Vol. XVII, U.S.Pharmacopoeia Convention, Inc., Rockville, Md. (1989), the relevantcontents of which is incorporated herein by reference.

The “semisolid gel” according to the current disclosure is a semisolid.The semisolid formulation apparent viscosity may increase withconcentration.

As used herein, “sequential administration” includes that theadministration of two agents (e.g., the compounds or compositionsdescribed herein) occurs separately on the same day or do not occur on asame day (e.g., occurs on consecutive days).

“Solution” according to the current disclosure may be a clear,homogeneous liquid dosage form that contains one or more chemicalsubstances dissolved in a solvent or mixture of mutually misciblesolvents. A solution is a liquid preparation that contains one or moredissolved chemical substances in a suitable solvent or mixture ofmutually miscible solvents. Because molecules of a drug substance insolution are uniformly dispersed, the use of solutions as dosage formsgenerally provides assurance of uniform dosage upon administration andgood accuracy when the solution is diluted or otherwise mixed.

The term “solvent,” as used herein, refers to a liquid solvent eitheraqueous or non-aqueous. The selection of the solvent depends notably onthe solubility of the composition on said solvent and on the mode ofadministration. Aqueous solvent may consist solely of water, or mayconsist of water plus one or more miscible solvents, and may containdissolved solutes such as sugars, buffers, salts or other excipients.The more commonly used non-aqueous solvents are the short-chain organicalcohols, such as, methanol, ethanol, propanol, short-chain ketones,such as acetone, and poly alcohols, such as glycerol. The solvent may bepresent in any suitable amount

By “subject” or “patient” is meant either a human or non-human animal,such as a mammal. “Subject” may include any animal, including horses,dogs, cats, pigs, goats, rabbits, hamsters, monkeys, guinea pigs, rats,mice, lizards, snakes, sheep, cattle, fish, and birds. A human subjectmay be referred to as a patient.

“Suspension” as used herein is a liquid dosage form that contains solidparticles dispersed in a liquid vehicle.

As used herein, the term “syndrome” may refer to a group of symptomsthat consistently occur together or a condition characterized by a setof associated symptoms. A syndrome (e.g., inflammatory bowel syndrome)may be a set of medical signs and symptoms that are correlated with eachother and often, are correlated with a specific disease. A disease, onthe other hand, may be a health condition that has a clearly definedreason behind it. A syndrome (from the Greek word meaning ‘runtogether’) however, may produce a number of symptoms without anidentifiable cause. They may suggest the possibility of an underlyingdisease or even the chances of developing a disease.

The terms “treat,” “treating” or “treatment,” and other grammaticalequivalents as used herein, include alleviating, abating, ameliorating,or preventing a disease, condition (e.g., inflammatory bowel disease) orsymptoms, preventing additional symptoms, ameliorating or preventing theunderlying metabolic causes of symptoms, inhibiting the disease orcondition, e.g., arresting the development of the disease or condition,relieving the disease or condition, causing regression of the disease orcondition, relieving a condition caused by the disease or condition, orstopping the symptoms of the disease or condition, and are intended toinclude prophylaxis. The terms further include achieving a therapeuticbenefit and/or a prophylactic benefit. By therapeutic benefit is meanteradication or amelioration of the underlying disorder being treated.Also, a therapeutic benefit is achieved with the eradication oramelioration of one or more of the physiological symptoms associatedwith the underlying disorder such that an improvement is observed in thepatient, notwithstanding that the patient may still be afflicted withthe underlying disorder.

As used herein, “viscosity” refers to a fluid's resistance to flow.Viscosity agents may be used herein and include, for example, polyvinylalcohol, polyvinyl pyrrolidone, methyl cellulose, hydroxy propylmethylcellulose, hydroxyethyl cellulose, carboxymethyl cellulose,hydroxy propyl cellulose, other agents known to those skilled in theart, or a combination thereof.

The term “weight percent” or “% (w/w)” refers to a percentage of acomponent in a solution that is calculated on the basis of weight forthe component and the solvent. For example, a 1% (w/w) solution of acomponent would have 1 g of the component dissolved in a 100 g ofsolvent. The term “volume percent” or “% (v/v)” refers to a percentageof a component in a solution that is calculated on the basis of volumefor the component and the solvent. For example, a 1% (v/v) solution of acomponent would have 1 ml of the component dissolved in a 100 ml ofsolvent. The term “weight/volume percent” or “% (w/v)” refers to apercentage of a component in a solution that is calculated on the basisof weight for the component and on the basis of volume for the solvent.For example, a 1.0% (w/v) solution of a component would have 1 g of thecomponent dissolved in a 100 ml of solvent

2. Compounds

As discussed above, one aspect of the present invention provides acompound represented by the following Formula 1, an optical isomerthereof, or a pharmaceutically acceptable salt thereof.

wherein: n is 0, 1, or 2; A is -a¹-, which is an amino acidindependently selected from the group consisting of alanine, (Ala, A),arginine (Arg, R), asparagine (Asn, N), aspartic acid (Asp, D), cysteine(Cys, C), glutamic acid (Glu, E), glutamine (Gln, Q), glycine (Gly, G),histidine (His, H), isoleucine (Ile, I), leucine (Leu, L), lysine (Lys,K), methionine (Met, M), phenylalanine (Phe, F), proline (Pro, P),serine (Ser, S), threonine (Thr, T), tryptophan (Trp, W), tyrosine (Tyr,Y), and valine (Val, V), both terminal ends of the amino acid beingcoupled to a carbonyl group or an amine group by an amide bond; and R¹is a straight chain or branched chain C₁₋₃₆ alkyl, a straight chain orbranched chain C₂₋₃₆ alkenyl including at least one double bond, or astraight chain or branched chain C₂₋₃₆ alkynyl including at least onetriple bond.

The term “compound of the present invention”, and equivalentexpressions, are meant to embrace the compound of the Formula ashereinbefore described, which expression includes the pharmaceuticallyacceptable salts, and the solvates, e.g., hydrates, and the solvates ofthe pharmaceutically acceptable salts where the context so permits.

In accordance with some embodiments of the invention, a¹ may be

and R¹ may be a straight chain or branched chain C₁₋₃₆ alkyl (SEQ ID NO:19).

Non-limiting examples of the compounds include the following compounds:

Compounds according to embodiments of the present invention areeffective for preventing or treating various diseases includinginflammatory indications, cancers, and ophthalmic indications. Moreparticularly, the compounds are effective for suppressing the expressionof cytokines and/or chemokines (e.g., G-CSF, IL-2, SCF, VEGF, CX3CL1,IGFBP5, IGFBP6, IL-1α, IL-1β, IL-6, IL-9, MCP-1, MIP-3αa, IL12p40/70,MIG, TNF-α, and VCAM-1). The compounds are also effective for inhibitingdecomposition of IκB in inflammation signaling pathway mediated by MyD88(myddosome complex) and/or RIP 1, thereby preventing NF-κB from beingtransported into nucleus of a cell. In addition, effective concentrationof the compounds in a targeted cell/tissue remains for a sufficienttime.

3. Preparation Methods

Another aspect of the present invention provides a method for preparingthe compound represented by Formula 1 (SEQ ID NO: 1). The method, asillustrated by the Reaction Scheme 1 shown below, comprises: reacting acompound 2 with a compound 3 to prepare a compound 4 (step 1);hydrolyzing the compound 4 in the presence of a base to prepare acompound 5 (step 2); reacting the compound 5 with a compound 6 toprepare a compound 7 (step 3); hydrolyzing the compound 7 in thepresence of a base to prepare a compound 8 (step 4); reacting thecompound 8 with a compound 9 to prepare a compound 10 (step 5);hydrolyzing the compound 10 in the presence of a base to prepare thecompound of Formula I (SEQ ID NO: 1) (step 6).

wherein A, R¹ and n are the same as defined in claim 1 and R² is astraight chain or branched chain C₁₋₅ alkyl.

In some embodiments, in the step 1, the compound 2 may be coupled withthe compound 3 in the presence of1-ethy-3-(3-dimethylaminopropyl)carbodiimide (EDCI),hydroxybenzotriazole (HOBt), and a base. The base can be an organic orinorganic base. Non-limiting examples of the organic base includepyridine, triethylamine (TEA), N,N-diisopropylethlyamine (DIPEA), and1,8-diazabicyclo[5.4.0]unde-7-ene (DBU). Non-limiting examples of theinorganic base include sodium hydroxide, sodium carbonate, potassiumcarbonate, cesium carbonate, and sodium hydride. These may be usedstoichiometric or excess, alone or in combination. Non-limiting examplesof the solvent that can be used to react the compound 2 with thecompound 3 include an ether (e.g., tetrahydrofuran (THF), dioxane, ethylether and 1,2-dimethoxyethane), an alcohol (e.g., methanol, ethanol,propanol, and butanol), dimethylformamide (DMF), dimethylsulfoxide(DMSO), dichloromethane (DCM), dichloroethane, water, acetone,benzenesulfonate, toluensulfonate, chlorobenzenesulfonate,xylenesulfonate, ethylacetate, phenylacetate, phenylpropionate,phenylbutyrate, citrate, lactate, hydroxybutyrate, glycolate, maleate,tartrate, methansulfonate, propanesulfonate, naphthalen-1-sulfonate,naphthalen-2-sulfonate, and mandelate. The solvent can be used alone orin combination.

The base in the step 2 can be an organic or inorganic base. Likewise,non-limiting examples of the organic base that can be used in the step 2include pyridine, triethylamine, N,N-diisopropylethlyamine (DIPEA), and1,8-diazabicyclo[5.4.0]unde-7-ene (DBU). Non-limiting examples of theinorganic base include sodium hydroxide, sodium carbonate, potassiumcarbonate, cesium carbonate, and sodium hydride. These may be usedstoichiometric or excess, alone or in combination. Non-limiting examplesof the solvent that can be used to react the compound 4 with thecompound 5 include an ether (e.g., tetrahydrofuran (THF), dioxane, ethylether and 1,2-dimethoxyethane), an alcohol (e.g., methanol, ethanol,propanol, and butanol), dimethylformamide (DMF), dimethylsulfoxide(DMSO), dichloromethane (DCM), dichloroethane, water, acetone,benzenesulfonate, toluensulfonate, chlorobenzenesulfonate,xylenesulfonate, ethylacetate, phenylacetate, phenylpropionate,phenylbutyrate, citrate, lactate, hydroxybutyrate, glycolate, mandelate,tartrate, methansulfonate, propanesulfonate, naphthalen-1-sulfonate,naphthalen-2-sulfonate, and mandelate. The solvent can be used alone orin combination.

The step 3 and the step 5 may be performed in the manner identical orsimilar to the step 1. The step 4 and the step 6 may be performed in themanner identical or similar to the step 2.

Preparation of Compound 2

Examples of the compound 2 represented by the following Formula 2, whichis the starting material of the Reaction Scheme 1, may be prepared by,e.g., the

Preparation Method A described below.

wherein n is 0, 1, or 2; A is -a¹-, which is an amino acid independentlyselected from the group consisting of alanine, (Ala, A), arginine (Arg,R), asparagine (Asn, N), aspartic acid (Asp, D), cysteine (Cys, C),glutamic acid (Glu, E), glutamine (Gln, Q), glycine (Gly, G), histidine(His, H), isoleucine (Ile, I), leucine (Leu, L), lysine (Lys, K),methionine (Met, M), phenylalanine (Phe, F), proline (Pro, P), serine(Ser, S), threonine (Thr, T), tryptophan (Trp, W), tyrosine (Tyr, Y),and valine (Val, V), both terminal ends of the amino acid being coupledto a carbonyl group or an amine group by an amide bond; and R¹ is astraight chain or branched chain C₁₋₃₆ alkyl, a straight chain orbranched chain C₂₋₃₆ alkenyl including at least one double bond, or astraight chain or branched chain C₂₋₃₆ alkynyl including at least onetriple bond

[Preparation Method A]

A compound represented by the Formula a shown below is coupled with anamino acid selected from the group consisting of alanine, (Ala, A),arginine (Arg, R), asparagine (Asn, N), aspartic acid (Asp, D), cysteine(Cys, C), glutamic acid (Glu, E), glutamine (Gln, Q), glycine (Gly, G),histidine (His, H), isoleucine (Ile, I), leucine (Leu, L), lysine (Lys,K), methionine (Met, M), phenylalanine (Phe, F), proline (Pro, P),serine (Ser, S), threonine (Thr, T), tryptophan (Trp, W), tyrosine (Tyr,Y), and valine (Val, V) in the presence of1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, hydroxybenzotriazole, anda base to form an amide bond, thereby preparing the compound 2.

(R¹ is same as defined in Formula 2).

4. Compositions/Formulations

A still another aspect of the present invention provides a compositionfor preventing, improving, and treating various diseases (e.g.,inflammatory indications, cancers, and ophthalmic indications), whichcomposition comprises, as an active component, at least one of thecompounds, at least one of the optical isomers, or at least one of thesalts.

Compositions in accordance with some embodiments may suppress expressionof cytokines and/or chemokines including G-CSF, IL-2, SCF, VEGF, CX3CL1,IGFBP5, IGFBP6, IL-1α,IL-1β, IL-6, IL-9, MCP-1, MIP-3α, IL12p40/70, MIG,TNF-α, and VCAM-1. Compositions in accordance with other embodiments maysuppress activity of NF-κB. Compositions in accordance with otherembodiments may inhibit formation of an inflammatory signal transductioncomplex mediated by MyD88. Compositions in accordance with otherembodiments may inhibit formation of an inflammatory signal transductioncomplex mediated by RIP1. Compositions in accordance with otherembodiments may inhibit formation of an inflammatory signal transductioncomplex mediated by Pellino-1.

In some embodiments, the present invention provides a composition forpreventing, improving, and treating inflammatory bowel disease(including closely related disorders), which comprises, as an activecomponent, at least one of the compounds, at least one of the opticalisomers, or at least one of the salts. The inflammatory bowel diseasemay include, but are not limited to, ulcerative colitis, Behcet'sdisease, and Crohn's disease. The composition may further comprise anadditive.

In some embodiments, the present invention provides a composition forpreventing, improving, or treating multiple sclerosis, psoriasis,sepsis, geographic atrophy, wet age-related macular disease, dryage-related macular disease, diabetic retinopathy, infectious lungdiseases, bacterial pneumonia, viral pneumonia, diffuse large B-celllymphoma, viral infection, autoimmune disease, blood cancer includinglymphoma, and tumors in internal organs, which comprises, as an activecomponent, at least one of the compounds, at least one of the opticalisomers, or at least one of the salts.

In some embodiments, the present invention provides a composition forpreventing, improving, or treating alopecia, which comprises, as anactive component, at least one of the compounds, at least one of theoptical isomers, or at least one of the salts, wherein the activecomponent inhibits expression of IL-6 in scalp and hair follicles.

The present invention embraces formulations suitable for theadministration of the compounds described herein. The compoundsdescribed herein can be in formulations (including pharmaceuticalcompositions) with additives such as excipients (e.g., one or moreexcipients), antioxidants (e.g., one or more antioxidants), stabilizers(e.g., one or more stabilizers), preservatives (e.g., one or morepreservatives), pH adjusting and/or buffering agents (e.g., one or morepH adjusting and/or buffering agents), tonicity adjusting agents (e.g.,one or more tonicity adjusting agents), thickening agents (e.g., one ormore thickening agents), suspending agents (e.g., one or more suspendingagents), binding agents (e.g., one or more binding agents),viscosity-increasing agents (e.g., one or more viscosity-increasingagents), and the like, provided that the additional components arepharmaceutically acceptable for the particular condition to be treated.In some embodiments, the formulation may include combinations of two ormore of the additional components as described herein (e.g., 2, 3, 4, 5,6, 7, 8, or more additional components). In some embodiments, theadditives include processing agents and drug delivery modifiers andenhancers, such as, for example, calcium phosphate, magnesium stearate,talc, monosaccharides, disaccharides, starch, gelatin, cellulose, methylcellulose, sodium carboxymethyl cellulose, dextrose,hydroxypropyl-beta-cyclodextrin, polyvinylpyrrolidinone, low meltingwaxes, ion exchange resins, and the like, as well as combinations of anytwo or more thereof. Other suitable pharmaceutically acceptableexcipients are described in “Remington's Pharmaceutical Sciences,” MackPub. Co., New Jersey (1991), and “Remington: The Science and Practice ofPharmacy,” Lippincott Williams & Wilkins, Philadelphia, 20th edition(2003) and 21st edition (2005), which are incorporated herein byreference.

Formulations of the pharmaceutical compositions appropriate foradministration by any medically acceptable means are included in theinvention. The pharmaceutical formulations may comprise apharmaceutically acceptable carrier appropriate to the means ofadministration and a pharmaceutically acceptable compound (composition).For example, formulations of the composition described herein may besuitable for oral administration. They can be formed in various formsincluding solutions, suspensions, semi-liquids, semi-solids, gels,emulsions, ointments, tablets, and creams. Tablet forms can include oneor more of lactose, sucrose, mannitol, sorbitol, calcium phosphates,corn starch, potato starch, microcrystalline cellulose, gelatin,colloidal silicon dioxide, talc, magnesium stearate, stearic acid, andother excipients, colorants, fillers, binders, diluents, bufferingagents, moistening agents, preservatives, flavoring agents, dyes,disintegrating agents, and pharmaceutically compatible carriers.

The compositions (formulations) may be administered via many routesincluding, not limited to, oral, nasal, pulmonary, rectal, buccal,vaginal, ocular, and transdermal routes. The mode, frequency, andeffective amount of administration of the compositions (formulations)can be decided according to methods known in the art and/or the methodsdescribed herein (e.g., oral administration, 0.1-1,000 mg/day, once aday). For example, they can be administered alone or in combination. Forexample, they can be concurrently administered, co-administered, and/orintermittently administered.

5. Methods of Using Compounds, Compositions, or Formulations A furtheraspect of the present invention provides a method for preventing,improving, or treating various diseases (e.g., inflammatory indications,cancers, and ophthalmic indications), which comprises administering to asubject in need the composition (or compound or formulation describedherein).

In an embodiment, the present invention provides a method forpreventing, improving, or treating inflammatory bowel disease, whichcomprises administering the composition (or compound or formulationdescribed herein) to a subject in need a composition containing, as anactive component, at least one of the compounds, at least one of theoptical isomers, or at least one of the salts.

In another embodiment, the prevent invention provides a method forpreventing, improving, or treating disease or syndrome, which methodcomprises administering to a subject in need a composition containing,as an active component, at least one of the compounds, at least one ofthe optical isomers, or at least one of the salts. The disease orsyndrome may involve formation of a Pellino-1 induced inflammatorysignal transduction complex containing MyD88, RIP1, or both. The diseaseor syndrome may include, but not limited to, multiple sclerosis,psoriasis, sepsis, geographic atrophy, wet age-related macular disease,dry age-related macular disease, diabetic retinopathy, infectious lungdiseases, bacterial pneumonia, viral pneumonia, diffuse large B-celllymphoma, viral infection, autoimmune disease, blood cancer includinglymphoma, and tumors in internal organs (e.g., liver, lung, intestine,prostate, pancreas and the like).

In still another embodiment, the prevent invention provides a method forpreventing, improving, or treating geographic atrophy, wet age-relatedmacular disease, dry age-related macular disease, or diabeticretinopathy, which method comprises administering to a subject in need acomposition containing, as an active component, at least one of thecompounds, at least one of the optical isomers, or at least one of thesalts. The compound(s), the optical isomer(s), and the salt(s) may havea pharmaceutical effect on retinal pigment epithelium cells. In retinalpigment epithelium cells, they may inhibit expression of at least oneprotein selected from the group consisting of Nox-4, VEGF, VEGFR1,VEGFR2, Ang2, EPO and EPOR. In retinal pigment epithelium cells, theymay increase expression of Ang 1, Tie2, or both.

EXAMPLES

The present invention will be explained in more detail with thefollowing examples. The examples are presented solely for the purpose ofillustration of the present invention and the present invention will notbe limited to the examples.

Example 1 Preparation of Compounds Example 1.1(S)-3-(4-hydroxyphenyl)-2-(2-((S)-1 -((S)-1-palmitoylpyrrolidine-2-carbonyl)pyrrolidine-2-carboxamido)acetamido)propanoicacid (Pal-PPGY-OH) (SEQ ID NO: 2)

Step 1: Preparation of (S)-methyl1-((S)-1-palmitoylpyrrolidine-2-carbonyl)pyroliddine-2-carboxylate

A mixture solution was made by mixing(S)-1-palmitoylpyrrolidine-2-carboxylic acid (10.0 g, 28.3 mmol)prepared in the step 2 of Example 1.2, EDCI (5.96 g, 31.1 mmol), HOBt(4.20 g, 31.1 mmol), and triethylamine (11.8 mL, 84.9 mmol) indichloromethane. Proline methyl ester hydrochloride (5.15 g, 31.1 mmol)was added to the mixture solution. The resulting mixture was agitated atroom temperature overnight, concentrated under reduced pressure, dilutedwith sodium bicarbonate aqueous solution, and extracted with ethylacetate three times. The whole organic layer was washed with salinesolution and washed with 1N HCl three times. The resultant was washedwith saline solution, dried with anhydrous magnesium sulfate, andconcentrated under reduced pressure to obtain (S)-methyl1-((S)-1-palmitoylpyrrolidine-2-carbonyl)pyrrolidine-2-carboxlate (11.4g, yield 87%).

¹H-NMR(300 MHz, CDCl₃) δ 4.69-4.65 (m, 1H), 4.54-4.58 (m, 1H), 3.83-3.93(m, 1H), 3.58-3.72 (m,5H), 3.45-3.53 (m, 1H), 1.89-2.31 (m, 10H),1.60-1.64 (m, 2H), 1.25 (m, 24H), 0.88 (t, J=6.87Hz, 3H).

MS (ESI), calcd for C₂₇H₄₈N₂O₄464.4, found m/z465.2 (M+H⁺).

Step 2: Preparation of (S)-1-((S)-1-palmitoylpyrrolidine-2-carbonyl)pyrrolidine-2-carboxylic acid (S)-methyl1-((S)-1-palmitoylpyrrolidine -2-carbonyl)pyrrolidine-2-carboxlate (15.0g, 32.3 mmol) prepared in the step 1 was mixed with tetrahydrofuran.Sodium hydroxide (2.58 g, 64.6 mmol) aqueous solution was added to themixture solution. The resulting mixture was agitated at room temperatureovernight and concentrated. 1N HCl was added to adjust the pH to 1.0.The aqueous layer thereof was extracted with ethyl acetate three times.The whole organic layer was dried with anhydrous magnesium sulfate andconcentrated to obtain(S)-1-((S)-1-palmitoylpyrrolidine-2-carbonyl)pyrrolidine-2-carboxylicacid (13.2 g, yield 91%) as white solid.

MS (ESI), calcd for C₂₆H₄₆N₂O4450.3, found m/z451.1 (M+H⁺).

Step 3: Preparation of ethyl2-((S)-1-((S)-1-palmitoylpyrrolidine-2-carbonyl)pyrrolidine-2-carboxamido)acetate

(S)-1-((S)-1-palmitoylpyrrolidine-2-carbonyl)pyrrolidine-2-carboxylicacid (12 g, 26.6 mmol) prepared in the step 2 was mixed withdichloromethane. Glycine ethyl ester hydrochloride (4.09 g, 29.3 mmol),EDCI (5.62 g, 29.3 mmol), HOBt (3.96 g, 29.3 mmol) and triethyamine(11.1 mL, 79.8 mmol) were added to the mixture solution. The resultingmixture was agitated at room temperature overnight, concentrated underreduced pressure, diluted with sodium carbonate aqueous solution, andextracted with ethyl acetate three times. The whole organic layer waswashed with saline solution and washed with 1N HCl three times. Theorganic layer was washed with saline solution, dried with anhydrousmagnesium sulfate, and concentrated to obtain ethyl2-((S)-1-((S)-1-palmitoylpyrrolidine-2-carbonyl)pyrrolidine-2-carboxyamido)acetate(11.1 g, yield 78%).

MS (ESI), calcd for C₃₀H₅₃N₃O₅535.4, found m/z536.5 (M+H⁺).

Step 4: Preparation of2-((S)-1-((S)-1-palmitoylpyrrolidine-2-carbonyl)pyrrolidine-2-carboxamido)aceticacid

Ethyl2-((S)-1-((S)-1-palmitoylpyrrolidine-2-carbonyl)pyrrolidine-2-carboxyamido)acetate(12 g, 22.4 mmol) prepared in the step 3 was mixed with tetrahydrofuran.Sodium hydroxide (1.79 g, 44.8 mmol) aqueous solution was added to themixture solution. The resulting mixture was agitated at room temperatureovernight and concentrated. 1N HCl was added to adjust the pH to 1.0.The aqueous layer was extracted with ethyl acetate three times. Thewhole organic layer was dried with anhydrous magnesium sulfate andconcentrated to obtain2-((S)-1-((S)-1-palmitoylpyrrolidine-2-carbonyl)pyrrolidine-2-carboxamido)aceticacid (9.5 g, yield 84%).

MS (ESI), calcd for C₂₈H₄₉N₃O₅507.4, found m/z508.2 (M+H⁺).

Step 5: Preparation of (S)-methyl3-(4-hydroxyphenyl)-2-(2-((S)-1-((S)-1-palmitoylpyrrolidine-2-carbonyl)pyrrolidine-2-carboxamido)acetamido)propanoate

2-((S)-1-((S)-1-palmitoylpyrrolidine-2-carbonyl)pyrrolidine-2-carboxamido)aceticacid (10 g, 19.7 mmol) prepared in the step 4 was mixed withdichloromethane. Tyrosine methyl ester (4.23 g, 21.7 mmol), EDCI (4.16g, 21.7 mmol), HOBt (21.7 g, 21.7 mmol), and triethylamine (8.19 mL,59.1 mmol) were added to the mixture solution. The resulting mixture wasagitated at room temperature overnight, concentrated under reducedpressure, diluted with sodium bicarbonate aqueous solution, andextracted with ethyl acetate three times. The whole organic layer waswashed with saline solution and washed with 1N HCl three times. Theorganic layer was washed with saline solution, dried with anhydrousmagnesium sulfate, concentrated, and purified with MPLC to obtain(S)-methyl3-(4-hydroxyphenyl)-2-(2-((S)-1-((S)-1-palmitoylpyrrolidine-2-carbonyl)pyrrolidine-2-carboxamido)acetamido)propanoate(8.4 g, yield 62%).

MS (ESI), calcd for C₃₇H₆₀N₄O₇684.4, found m/z685.2 (M+H⁺).

Step 6: Preparation of(S)-3-(4-hydroxyphenyl)-2-(2-((S)-1-((S)-1-palmitoylpyrrolidine-2-carbonyl)pyrrolidine-2-carboxamido)acetamido)propanoicacid

(S)-methyl3-(4-hydroxyphenyl)-2-(2-((S)-1-((S)-1-palmitoylpyrrolidine-2-carbonyl)pyrrolidine-2-carboxamido)acetamido)propanoate(4.9 g, 7.16 mmol) prepared in the step 5 was mixed withtetrahydrofuran. Sodium hydroxide (0.86 g, 21.5 mmol) aqueous solutionwas added to the mixture solution. The resulting mixture was agitated atroom temperature overnight and concentrated. 1N HCl was added to adjustthe pH to 1.0. The aqueous layer was extracted with ethyl acetate threetimes. The whole organic layer was dried with anhydrous magnesiumsulfate and concentrated to obtain(S)-3-(4-hydroxyphenyl)-2-(2-((S)-1-((S)-1-palmitoylpyrrolidine-2-carbonyl)pyrrolidine-2-carboxamido)acetamido)propanoicacid (SEQ ID NO: 2) (4.5 g, yield 93%).

¹H-NMR(300 MHz, MeOD) δ 7.04 (d, J=8.31 Hz, 2H) 6.70 (d, J=8.37 Hz,2H),4.38-4.68 (m,3H), 3.44-4.04 (m, 6H), 2.91-3.13 (m, 2H), 1.81-2.38 (m,10H), 1.54-1.60 (m, 2H), 1.30 (m, 24H), 0.88 (t, J=6.63 Hz, 3H).

MS (ESI), calcd for C₃₇H₅₈N₄O₇670.4, found m/z671.3 (M+H⁺).

Example 1.2(S)-3-(4-hydroxyphenyl)-2-(2((S)-1-palmitoylpyrrolidine-2-carboxamido)acetamido)propanoicacid(Pal-PGY-OH)

Step 1: (S)-methyl 1-palmitoylpyrrolidine-2-carboxylate

Palmitic acid (7 g, 27.3 mmol), EDCI (5.78 g, 30.0 mmol), HOBt (4.05 g,30.0 mmol), and, triethylamine (11.4 mL, 81.9 mmol) were mixed withdichloromethane. Proline methyl ester hydrochloride (4.97 g, 30.0 mmol)was added to the mixture solution. The resulting mixture was agitated atroom temperature overnight, concentrated under reduced pressure, dilutedwith sodium carbonate aqueous solution, and extracted with ethyl acetatethree times. The whole organic layer was washed with saline solution andwashed with 1N HCl three times. The organic layer was washed with salinesolution, dried with anhydrous magnesium sulfate, and concentrated underreduced pressure to obtain (S)-methyl1-palmitoylpyrrolidine-2-carboxylate (9.6 g, yield 96%) as viscousliquid.

¹H-NMR(300 MHz, CDCl₃) δ 4.46-4.50 (m, 1H), 3.47-3.75 (m,5H), 1.90-2.36(m, 6H), 1.59-1.69 (m, 2H), 1.25(m, 24H), 0.88 (t, J=6.84 Hz, 3H)

MS (ESI), calcd for C₂₂H₄₁NO₃367.3, found m/z368 (M+H⁺).

Step 2: Preparation of (S)-1-palmitoylpyrrolidine-2-carboxylic acid

(S)-methyl 1-palmitoylpyrrolidine-2-carboxylate (10.0 g, 27.2 mmol)prepared in the step 1 was mixed with tetrahydrofuran. Sodium hydroxide(3.26 g, 81.6 mmol) aqueous solution was added to the mixture solution.The resulting mixture was agitated at room temperature overnight andconcentrated. 1N HCl was added to adjust the pH to 1.0. The aqueouslayer was extracted with ethyl acetate three times. The whole organiclayer was dried with anhydrous magnesium sulfate and concentrated toobtain (S)-1-palmitoylpyrrolidine-2-carboxylic acid (8.6 g, yield 89%)as white solid.

¹H-NMR(300 MHz, CDCl₃) δ 4.59-62 (m, 1H), 3.42-3.59 (m,2H), 2.46-2.53(m, 1 H), 2.33-2.38 (m, 2H), 1.93-2.01 (m, 3H), 1.62-1.69 (m, 2H), 1.25(m, 24H), 0.88 (t, J=6.90 Hz, 3H)

MS (ESI), calcd for C₂₁H₃₉NO₃353.3, found m/z354.2 (M+H⁺).

Step 3: preparation of (S)-ethyl2-(1-palmitoylpyrrolidine-2-carboxamido)acetate

(S)-1-palmitoylpyrrolidine-2-carboxylic acid (10 g, 28.3 mmol) preparedin the step 2, glycine ethyl ester hydrochloride (4.34 g, 31.1 mmol),EDCI (5.76 g,31.1 mmol), HOBt (4.20 g,31.1 mmol), and triethyamine(1,5.7 mL, 113 mmol) were mixed with dichloromethane. The resultingmixture was agitated at room temperature overnight, concentrated underreduced pressure, diluted with sodium carbonate aqueous solution, andextracted with ethyl acetate three times. The whole organic layer waswashed with saline solution and washed with 1N HCl three times. Theorganic layer was washed with saline solution, dried with anhydrousmagnesium sulfate, and concentrated to obtain (S)-ethyl2-(1-palmitoylpyrrolidine-2-carboxamido)acetate (10.7 g, yield 86%).

MS (ESI), calcd for C₂₅H₄₆N₂O₄438.3, found m/z439.1 (M+H⁺).

Step 4: Preparation of(S)-2-(1-palmitoylpyrrolidine-2-carboxamido)acetic acid

(S)-ethyl 2-(1-palmitoylpyrrolidine-2-carboxamido)acetate (12 g, 27.4mmol) prepared in the step 3 was mixed with tetrahydrofuran. Sodiumhydroxide (2.20 g, 54.7 mmol) aqueous solution was added to the mixturesolution. The resulting mixture was agitated at room temperatureovernight and concentrated. 1N HCl was added to adjust the pH to 1.0.The aqueous layer was extracted with ethyl acetate three times. Thewhole organic layer was dried with anhydrous magnesium sulfate andconcentrated to obtain(S)-2-(1-palmitoylpyrrolidine-2-carboxamido)acetic acid (10.2 g, yield91%) as white solid.

MS (ESI), calcd for C₂₃H₄₂N₂O₄410.3, found m/z 411.3 (M+H⁺).

Step 5: Preparation of (S)-methyl3-(4-hydroxyphenyl)-2-(2-((S)-1-palmitoylpyrrolidine-2-carboxamido)acetamido)propanoate

(S)-2-(1-palmitoylpyrrolidine-2-carboxamido)acetic acid (7 g, 27.3 mmol)prepared in the step 4 was mixed with dichloromethane. Tyrosine methylester (5.86 g, 30.0 mmol), EDCI(5.78 g, 30.0 mmol), HOBt(4.05 g, 30.0mmol), and triethylamine (11.4 mL, 81.9 mmol) were added to the mixturesolution. The resulting mixture was agitated at room temperatureovernight, concentrated under reduced pressure, diluted with sodiumbicarbonate aqueous solution, and extracted with ethyl acetate threetimes. The whole organic layer was washed with saline solution andwashed with 1N HCl three times. The organic layer was washed with salinesolution, dried with anhydrous magnesium sulfate, concentrated, andpurified with MPLC to obtain (S)-methyl3-(4-hydroxyphenyl)-2-(2-((S)-1-palmitoylpyrrolidine-2-carboxamido)acetamido)propanoate (9.8 g, yield 61%).

¹H-NMR(300 MHz, CDCl₃) δ 7.25-7.50 (m, 3H), 6.93 (d, J=8.34 Hz, 2H) 6.70(d, J=8.34 Hz,2H), 4.70-4.77 (m,1H), 4.39-4.43 (m, 1H), 3.95-4.21 (m,1H), 3.41-3.72 (m, 5H), 2.92-3.12 (m, 2H), 1.91-2.35 (m, 7H), 1.57-1.61(m, 2H), 1.25 (m, 24H), 0.88 (t, J=6.87 Hz, 3H)

MS (ESI), calcd for C₃₃H₅₃N₃O₆587.4, found m/z588.1 (M+H⁺).

Step 6: Preparation of(S)-3-(4-hydroxyphenyl)-2-(2-((S)-1-palmitoylpyrrolidine-2-carboxamido)acetamido)propanoic acid

(S)-methyl 3-(4-hydroxyphenyl)-2-(2-((S)-1-palmitoylpyrrolidine-2-carboxamido)acetamido)propanoate (2.85 g, 4.85 mmol) prepared in thestep 5 was mixed with tetrahydrofuran. Sodium hydroxide (0.58 g, 14.6mmol) aqueous solution was added to the mixture solution. The resultingmixture was agitated at room temperature overnight and concentrated. 1NHCl was added to adjust the pH to 1.0. The aqueous layer was extractedwith ethyl acetate three times. The whole organic layer was dried withanhydrous magnesium sulfate and concentrated to obtain(S)-3-(4-hydroxyphenyl)-2-(2-((S)-1-palmitoylpyrrolidine-2-carboxamido)acetamido)propanoic acid (2.2 g, yield 79%) as whitesolid.

¹H-NMR(300 MHz, MeOD) δ 7.03 (d, J=8.40 Hz,2H) 6.70 (d, J=8.40 Hz,2H),4.58-4.61 (m,1H), 4.33-4.56 (m, 1H), 3.58-4.37 (m, 4H), 2.96-3.15 (m,2H), 1.92-2.39 (m, 6H), 1.55-1.62 (m, 2H), 1.29 (m, 24H), 0.91 (t,J=6.87 Hz, 3H) MS (ESI), calcd for C₃2H₅1N₃O₆573.4, foundm/z574.2(M+H⁺).

Example 1.3 Palmitoyl-L-alanyl-L-prolylglycyl-L-tyrosine(pal-APGY-OH)(SEQ ID NO: 3)

The compound was prepared according to the following Reaction Scheme 2.

The compound (1) (10 g, 46.5 mmol), the compound (2) (7.15 g, 51.2mmol), EDCI.HCl (9.82 g, 51.2 mmol), HOBt (6.92 g, 51.2 mmol), andtriethylamine (19.4 mL, 140 mmol) were mixed with dichloromethane. Theresulting mixture was agitated at room temperature overnight,concentrated under reduced pressure, diluted with sodium carbonateaqueous solution, and extracted with ethyl acetate three times. Thewhole organic layer was washed with saline solution and washed with 1NHCl three times. The organic layer was washed with saline solution,dried with anhydrous magnesium sulfate, and concentrated under reducedpressure to obtain the compound (3) (yield 91%) as viscous liquid.

LC-MS (ESI): calcd for C₁₄H₂₄N₂O₅300.2, found m/z 301.2 (M+H⁺).

The compound (3) (12 g, 40 mmol) was mixed with tetrahydrofuran. Sodiumhydroxide (6.40 g, 160 mmol) aqueous solution was added to the mixturesolution. The resulting mixture was agitated at room temperatureovernight and concentrated. 1N HCl was added to adjust the pH to 1.0.The aqueous layer was extracted with ethyl acetate three times. Thewhole organic layer was dried with anhydrous magnesium sulfate andconcentrated to obtain the compound (4) (yield 96%) as white solid.

LC-MS (ESI): calcd for C₁₂H₂₀N₂O₅ 272.1, found m/z 273.1 (M+H⁺).

The compound (4) (6.25 g, 23 mmol), the compound (5) (4.85 g, 25.3mmol), EDCI.HCl (4.85 g, 25.3 mmol), HOBt (43.42 g, 25.3 mmol), andtriethylamine (TEA, 12.8 mL, 96 mmol) were mixed with dichloromethane.The resulting mixture was agitated at room temperature overnight,concentrated under reduced pressure, diluted with sodium carbonateaqueous solution, and extracted with ethyl acetate three times. Thewhole organic layer was washed with sodiumbicarbonate aqueous solutiontwice, washed with saline solution, and washed with 1N HCl three times.The organic layer was washed with saline solution, dried with anhydrousmagnesium sulfate, and concentrated to obtain the compound (6) (yield87%) as white solid.

LC-MS (ESI): calcd for C₂₂H₃₁N₃O₇ 449.2, found m/z 450.2 (M+H⁺).

The compound (6) (8 g, 17.8 mmol) was dissolved in ethyl acetate. Anexcess amount of 4 N HCl in dioxane was added at room temperature. Theresulting mixture was agitated at room temperature for 4 hours andconcentrated under reduced pressure to obtain the compound (7) as whitesolid.

LC-MS (ESI): calcd for C₁₇H₂₃N₃O₅ 349.2, found m/z 350.2 (M+H⁺).

The compound (7) (0.25 g, 0.65 mmol), the compound (8) (Boc-alanine,0.12 g, 0.65 mmol), EDCI.HCl (0.25 g, 1.30 mmol), HOBt (0.18 g, 1.30mmol), and triethylamine (0.36 mL, 2.60 mmol) were mixed withdichlromethane. The resulting mixture was agitated at room temperatureovernight, concentrated under reduced pressure, diluted with sodiumcarbonate aqueous solution, and extracted with ethyl acetate threetimes. The whole organic layer was washed with sodium bicarbonateaqueous solution twice, washed with saline solution, and washed with 1NHCl three times. The organic layer was washed with saline solution,dried with anhydrous magnesium sulfate, concentrated, and purified byusing MPLC (dichloromethane/2-propanol) to obtain the compound (9)(yield 3%).

LC-MS (ESI): calcd for C₂₅H₃₆N₄O₈ 520.3, found m/z 520.7 (M+H⁺).

The compound (9) (0.11 g, 0.21 mmol) was dissolved in ethyl acetate. Anexcess amount of 4 N HCl in dioxane was added at room temperature andagitated at room temperature for 4 hours. The resulting mixture wasconcentrated under reduced pressure to obtain the compound (10) as whitesolid.

LC-MS (ESI): calcd for C₂₀H₂₈N₄O₆ 420.2, found m/z 420.6 (M+H⁺).

To a solution of palmitic acid (0.02 g, 0.08 mmol) in dichloromethanewas added compound (10) (0.04 g, 0.09 mmol), EDCI.HCl (0.03 g, 0.16mmol), HOBt (0.02 g, 0.16 mmol), and triethylamine (0.04 mL, 0.32 mmol).The resultant was was agitated at room temperature overnight,concentrated under reduced pressure, diluted with sodium carbonateaqueous solution, and extracted with ethyl acetate three times. Thewhole organic layer was washed with saline solution, and washed with 1NHCl three times. The organic layer was washed with saline solution,dried with anhydrous magnesium sulfate, concentrated, and purified byusing MPLC (dichloromethane/2-propanol) to obtain the compound (11)(yield 58%).

LC-MS (ESI): calcd for C₃₆H₅₈N₄O₇ 658.4, found m/z 659.1 (M+H⁺)

The compound (11) (0.03 g, 0.05 mmol) was mixed with tetrahydrofuran.Sodium hydroxide (0.008 g, 0.20 mmol) aqueous solution was added. Theresulting mixture was agitated at room temperature overnight andconcentrated. 1N HCl was added to adjust the pH to 1.0. The aqueouslayer was extracted with ethyl acetate three times. The whole organiclayer was dried with anhydrous magnesium sulfate and concentrated toobtain the compound (12) (yield 93%) as white solid.

¹H NMR (500 MHz, CD₃OD) δ 7.05 (d, J=8.50 Hz, 2H), 6.70 (d, J=8.50 Hz, 2H), 4.59-4.65 (m, 2H), 4.39-4.41 (m, 1H), 3.96-3.99 (m, 1H), 3.84-3.89(m, 1H), 3.65-3.76 (m, 2H), 3.10-3.14 (m, 1H), 2.97-3.01 (m, 1H),2.20-2.24 (m, 3H), 2.09-2.14 (m, 1H), 1.96-2.03 (m, 2H), 1.58-1.60 (m,3H), 1.31-1.36 (m, 30H), 0.92 (t, J=7.15 Hz, 3 H). LC-MS (ESI): calcdfor C₃₅H₅₆N₄O 7 644.4, found m/z 644.6 (M+H⁺).

Compounds of Examples 1.4 through 1.15 were prepared in the same manneras described in Example 1.3, using the compound (7) as the startingmaterial. NMR data of those compounds are shown below.

Example 1.4

Palmitoylglycyl-L-prolylglycyl-L-tyrosine (pal-GPGY-OH)(SEQ ID NO: 4) ¹HNMR (500 MHz, CD3OD) δ 7.03 (d, J=8.50 Hz, 2H), 6.70 (d, J=8.50 Hz, 2H),4.58-4.63 (m, 1H), 4.39-4.42 (m, 1H), 3.90-4.08 (m, 4H), 3.59-3.74 (m,3H), 3.09-3.12 (m, 1H), 2.96-3.00 (m, 1H), 1.99-2.31 (m, 7H), 1.56-1.66(m, 3H), 1.24-1.35 (m, 26H), 0.92 (t, J=7.05 Hz, 3H). LC-MS (ESI): calcdfor C₃4H₅4N₄O₇ 630.4, found m/z 630.8 (M+H⁺).

Example 1.5

((9Z,12Z)-octadeca-9,12-dienoyl)glycyl-L-prolylglycyl-L-tyrosine(linoleyl-GPGY-OH)(SEQ ID NO: 5) ¹H NMR (500 MHz, CD₃OD) δ 7.03 (d,J=8.50 Hz, 2H), 6.71 (d, J=8.50 Hz, 2H), 5.31-5.41 (m, 4H), 4.58-4.62(m, 1H), 4.39-4.42 (m, 1H), 3.99-4.08 (m, 2H), 3.70-3.73 (m, 1H),3.59-3.67 (m, 1H), 3.09-3.12 (m, 1H), 2.96-3.00 (m, 1H), 2.78-2.79 (m,3H), 2.25-2.31 (m, 1H), 2.18-2.22 (m, 2H), 1.99-2.13 (m, 7H), 1.56-1.64(m, 3H), 1.31-1.42 (m, 18H), 0.93 (t, J=7.10 Hz, 3H). LC-MS (ESI): calcdfor C₃₆H₅N₄O₇ 654.4, found m/z 655 (M+H⁺).

Example 1.6

Palmitoyl-L-phenylalanyl-L-prolylglycyl-L-tyrosine (pal-FPGY-OH)(SEQ IDNO: 6) ¹H NMR (500 MHz, CD₃OD) δ 7.27-7.28 (m, 5H), 7.07 (d, J=8.45 Hz,2H), 6.71 (d, J=8.30 Hz, 2H), 4.59-4.63 (m, 1H), 4.40-4.42 (m, 1H),3.99-4.02 (m, 1H), 3.85-3.89 (m, 1H), 3.73-3.78 (m, 1H), 3.52-3.55 (m,1H), 3.09-3.16 (m, 2H), 2.86-3.02 (m, 3H), 1.95-2.22 (m, 8H), 1.44-1.62(m, 4H), 1.31 (m, 25H), 0.92 (t, J=7.10 Hz, 3H). LC-MS (ESI): calcd forC₄₁H₆₀N₄O₇ 720.4, found m/z 721.1 (M+Hf).

Example 1.7

Hexanoyl-L-prolyl-L-prolylglycyl-L-tyrosine (hexanoyl-PPGY-OH)(SEQ IDNO: 7) ¹H NMR (500 MHz, CD₃OD) δ 7.05 (d, J=8.50 Hz, 2H), 6.70 (d,J=8.50 Hz, 2H), 4.66-4.68 (m, 1H), 4.54-4.57 (m, 1H), 4.40-4.44 (m, 1H),3.96-4.00 (m, 1H), 3.85-3.89 (m, 1H), 3.73-3.76 (m, 1H), 3.51-3.68 (m,3H), 3.08-3.12 (m, 1H), 2.97-3.02 (m, 1H), 2.31-2.41 (m, 2H), 2.20-2.29(m, 2H), 1.92-2.12 (m, 5H), (m, 8H), 1.57-1.66 (m, 3H), 1.31-1.38 (m,5H), 0.93 (t, J=7.00 Hz, 3H). LC-MS (ESI): calcd for C₂₇H₃₈N₄O₇ 530.3,found m/z530.7 (M+H⁺).

Example 1.8

Octanoyl-L-prolyl-L-prolylglycyl-L-tyrosine (octanoyl-PPGY-OH)(SEQ IDNO: 8) ¹H NMR (500 MHz, CD₃OD) δ 7.05 (d, J=8.50 Hz, 2H), 6.71 (d,J=8.50 Hz, 2H), 4.66-4.68 (m, 1H), 4.54-4.57 (m, 1H), 4.40-4.42 (m, 1H),3.96-4.00 (m, 1H), 3.85-3.89 (m, 1H), 3.73-3.77 (m, 1H), 3.51-3.68 (m,3H), 3.08-3.12 (m, 1H), 2.97-3.02 (m, 1H), 2.18-2.34 (m, 4H), 1.92-2.12(m, 5H), 1.57-1.61 (m, 2H), 1.32-1.35 (m, 10H), 0.92 (t, J=7.00 Hz, 3H).LC-MS (ESI): calcd for C₂₉H₄₂N₄O₇ 558.3, found m/z558.5 (M+H⁺).

Example 1.9

Decanoyl-L-prolyl-L-prolylglycyl-L-tyrosine (decanoyl-PPGY-OH)(SEQ IDNO: 9) ¹H NMR (500 MHz, CD₃OD) δ 7.03 (d, J=8.55 Hz, 2H), 6.71 (d,J=8.40 Hz, 2H), 4.66-4.68 (m, 1H), 4.54-4.57 (m, 1H), 4.40-4.42 (m, 1H),3.96-4.00 (m, 1H), 3.85-3.89 (m, 1H), 3.73-3.77 (m, 1H), 3.51-3.68 (m,3H), 3.08-3.12 (m, 1H), 2.97-3.02 (m, 1H), 2.18-2.39 (m, 5H), 1.92-2.14(m, 6H), 1.57-1.61 (m, 2H), 1.32-1.36 (m, 14H), 0.92 (t, J=7.15 Hz, 3H).LC-MS (ESI): calcd for C₃₁H₄₆N₄O₇ 586.3, found m/z 586.8 (M+H⁺).

Example 1.10

Stearoyl-L-prolyl-L-prolylglycyl-L-tyrosine (stearoyl-PPGY-OH)(SEQ IDNO: 10) ¹H NMR (500 MHz, CD₃OD) δ 7.03 (d, J=8.55 Hz, 2H), 6.71 (d,J=8.25 Hz, 2H), 4.66-4.68 (m, 1H), 4.55-4.57 (m, 1H), 4.40-4.44 (m, 1H),3.96-4.02 (m, 1H), 3.84-3.89 (m, 1H), 3.71-3.76 (m, 1H), 3.48-3.68 (m,4H), 3.08-3.12 (m, 1H), 2.97-3.02 (m, 1H), 2.14-2.42 (m, 5H), 1.90-2.14(m, 6H), 1.57-1.61 (m, 2H), 1.32-1.35 (m, 30H), 0.92 (t, J=7.15 Hz, 3H).LC-MS (ESI): calcd for C₃₉H₆₂N₄O₇ 698.5, found m/z 698.5 (M+H⁺).

Example 1.11

Hex-5-enoyl-L-prolyl-L-prolylglycyl-L-tyrosine (5-hexenoyl-PPGY-OH)(SEQID NO: 11) ¹H NMR (500 MHz, CD₃OD) δ 7.05 (d, J=8.55 Hz, 2H), 6.70 (d,J=8.25 Hz, 2H), 5.78-5.89 (m, 1H), 4.98-5.08 (m, 3H), 4.66-4.69 (m, 1H),4.54-4.57 (m, 1H), 4.40-4.44 (m, 1H), 3.97-4.01 (m, 1H), 3.84-3.90 (m,1H), 3.73-3.76 (m, 1H), 3.47-3.68 (m, 4H), 3.08-3.12 (m, 1H), 2.97-3.01(m, 1H), 2.33-2.42 (m, 2H), 2.20-2.30 (m, 2H), 2.06-2.15 (m, 4H),1.92-2.04 (m, 4H), 1.67-1.76 (m, 3H). LC-MS (ESI): calcd for C₂₇H₃₆N₄O₇528.3, found m/z 529 (M+H⁺).

Example 1.12

Oleoyl-L-prolyl-L-prolylglycyl-L-tyrosine (oleyl-PPGY-OH)(SEQ ID NO: 12)¹H NMR (500 MHz, CD₃OD) 6 7.03 (d, J=8.50 Hz, 2H), 6.71 (d, J=8.50 Hz,2H), 5.34-5.38 (m, 2H), 4.66-4.68 (m, 1H), 4.55-4.57 (m, 1H), 4.40-4.42(m, 1H), 3.96-4.01 (m, 1H), 3.84-3.90 (m, 1H), 3.73-3.77 (m, 1H),3.53-3.69 (m, 4H), 3.08-3.12 (m, 1H), 2.97-3.01 (m, 1H), 2.18-2.39 (m,5H), 2.06-2.12 (m, 2H), 1.92-2.05 (m, 3H), 1.58-1.61 (m, 3H), 1.31-1.35(m, 25H), 0.92 (t, J=7.0 Hz, 3H). LC-MS (ESI): calcd for C₃₉H₆₀N₄O₇696.4, found m/z 697.3 (M+H⁺).

Example 1.13

((9Z,12Z)-octadeca-9,12-dienoyl)-L-prolyl-L-prolylglycyl-L-tyrosine(linoleyl-PPGY-OH)(SEQ ID NO: 13) ¹H NMR (500 MHz, CD₃OD) δ 7.05 (d,J=8.45 Hz, 2H), 6.71 (d, J=8.45 Hz, 2H), 5.32-5.41 (m, 4H), 4.66-4.68(m, 1H), 4.55-4.57 (m, 1H), 4.40-4.43 (m, 1H), 3.96-4.01 (m, 1H),3.84-3.89 (m, 1H), 3.73-3.77 (m, 1H), 3.53-3.68 (m, 4H), 3.08-3.12 (m,1H), 2.97-3.01 (m, 1H), 2.80 (t, J=6.40 Hz, 2 H), 2.19-2.39 (m, 5H),1.93-2.13 (m, 10H), 1.59-1.61 (m, 3H), 1.31-1.40 (m, 15H), 0.92 (t,J=6.59 Hz, 3H). LC-MS (ESI): calcd for C₃₉H₅₈N₄O₇ 694.4, found m/z695.2(M +H⁺).

Example 1.14

Palmitoyl-L-valeryl-L-prolyl-L-prolylglycyl-L-tyrosine(Pal-VPPGY-OH)(SEQ ID NO: 16) ¹H NMR (500 MHz, CD₃OD) 6 7.04 (d, J=8.35Hz, 2H), 6.71 (d, J=8.35 Hz, 2H), 4.67-4.70 (m, 1H), 4.52-4.58 (m, 1H),4.39-4.43 (m, 1H), 3.77-4.05 (m, 4H), 3.64-3.73 (m, 2H), 3.49-3.60 (m,1H), 3.01-3.11 (m, 2H), 1.87-2.31 (m, 15H), 1.61-1.63 (m, 3H), 1.31 (m,24H), 1.01 (d, J=6.58 Hz, 3H), 0.98 (d, J=6.58 Hz, 3H), 0.92 (t, J=6.96Hz, 3H). LC-MS (ESI), calcd for C₄₂H₆₇N₅O₈ 769.5, found m/z 770.7(M+H⁺).

Example 1.15

Decanoyl-L-valeryl-L-prolyl-L-prolylglycyl-L-tyrosine(decanoyl-VPPGY-OH)(SEQ ID NO: 17) ¹H NMR (500 MHz, CD₃OD) δ 7.05 (d,J=8.45 Hz, 2H), 6.71 (d, J=8.45 Hz, 2H), 4.67-4.70 (m, 1H), 4.50-4.57(m, 1H), 4.39-4.43 (m, 1H), 3.95-4.05 (m, 2H), 3.80-3.91 (m, 1H),3.65-3.70 (m, 2H), 3.64-3.73 (m, 2H), 3.53-3.57 (m, 1H), 3.01-3.10 (m,2H), 1.89-2.31 (m, 12H), 1.61-1.63 (m, 3H), 1.31 (m, 24H), 1.01 (d,J=6.68 Hz, 3H), 0.97 (d, J=6.55 Hz, 3H), 0.92 (t, J=7.05 Hz, 3H). LC-MS(ESI), calcd for C₃₆H₅₅N₅O₈ 685.4, found m/z686.6 (M+H⁺).

Table 1 depicts the compounds according to Examples 1.1 through 1.15.

TABLE 1 Compounds according to Examples 1.1 through 1.15 Example No.(Compound No.) Chemical Formula 1.1 SEQ ID NO: 2

1.2

1.3 SEQ ID NO: 3

1.4 SEQ ID NO: 4

1.5 linoleyl-GPGY-OH SEQ ID NO: 5 1.6 SEQ ID NO: 6

1.7 hexanoyl-PPGY-OH SEQ ID NO: 7 1.8 octanoyl-PPGY-OH SEQ ID NO: 8 1.9SEQ ID NO: 9

 1.10 SEQ ID NO: 10

 1.11 SEQ ID NO: 11

 1.12 SEQ ID NO: 12

 1.13 SEQ ID NO: 13

 1.14 SEQ ID NO: 16

 1.15 SEQ ID NO: 17

EXAMPLE 2 ASSAYS Example 2.1 Suppression of Expression of IL-6

To evaluate suppression of expression of IL-6 by the compounds of thepresent invention, the following experiment was performed.

First, RAW 264.7 macrophage cells were purchased from American TypeCulture Collection (ATCC; Manassas, Va.), and were culture at atemperature of 37° C., under 5% CO₂ atmosphere, using 10% Fetal BovineSerum (FBS) and Dulbecco's Modified Eagle's Medium (DMEM) containing 1%penicillin/streptomycin.

The cultured RAW 264.7 macrophage cells were divided into a 6-well cellculture plate. After 24 hours, the cells were pretreated with compound1.1 (SEQ ID NO: 2), compound 1.2, and smaducin-6, respectively, in aconcentration of 100 nM for 30 min, and further treated withlipopolysaccharides (LPS) for two hours. These cells were collectedusing TRIzol® and the RNA was extracted. From 2 μg of the extracted RNA,cDNAs (complementary deoxyribonucleic acids) were synthesized andreverse transcription polymerase chain reactions (RT-PCR) and real-timepolymerase chain reactions (Real-Time PCR) were performed. Samples fromthe RT-PCR were confirmed by electrophoresis on agarose gels andquantified using densitometer. A glyceraldehyde 3-phosphatedehydrogenase (GAPDH) gene was used as a loading control for theinterleukin-6 gene.

FIG. 1A shows images depicting suppression of expression ofinterleukin-6 by compound 1.1 (SEQ ID NO: 2) and compound 1.2, and FIG.1B is a graph showing the quantified suppression of expression ofinterleukin-6 by the compound 1.1(SEQ ID NO: 2) and compound 1.2. Ascompared to the untreated sample, when the cells treated with thecompound according to the present invention, the expression ofinterleukin-6, which was induced by LPS treatment, was suppressed.

In addition, RAW 264.7 macrophage cells cultured as described above werealso divided into a 6-well plate, after 24 hours, were pretreated with100 nM of compound 1.1 (SEQ ID NO: 2) for 30 minutes, and furthertreated with LPS for two hours. The above cell culture was collectedand, using a cytokine array (Mouse cytokine array C3, RayBiotech),changes in amounts of cytokine and chemokine were quantified by adensitometer. The cytokines and chemokines detected by the compound ofthe present invention included G-CSF, IL-2, SCF, VEGF, CX3CL1, IGFBP5,IGFBP6, IL-1α, IL-1β, IL-6, IL-9, MCP-1, MIP-3α, IL12p40/70, MIG, TNF-α,and VCAM-1. As compared to the untreated samples, when the cells weretreated with the compounds of the present invention, the expression ofthe above cytokines and chemokines, which were induced by LPS treatment,were suppressed as statistically meaningful (FIGS. 2A-2D).

RAW 264.7 macrophage cells were treated with varying concentrations ofcompound 1.1 (SEQ ID NO: 2) from 50 pM to 500 nM for 30 minutes, andtreated with100 ng/mL of LPS for two hours. The induced expression ofinterleukin-6 was quantified as described above and the results werepresented in FIG. 3. In the RAW 264.7 macrophage cells, theconcentration of compound 1.1 (SEQ ID NO: 2) where the expression ofinterleukin-6 was suppressed to 50% or below (i.e. IC₅₀) was about 1.6nM.

Example 2.2 Suppression of Activity of NF-κ13

To evaluate whether NF-κB signal induced by LPS was specificallysuppressed by the compounds of the present invention, the followingexperiments were performed.

Specifically, 5x NF-κB-Luc reporter plasmid was transfected into RAW264.7 cells using Effectene (Qiagen, USA). The transfected cells werepretreated with 100 nM of compound 1.1 (SEQ ID NO: 2) for 30 minutes,and further treated with LPS (100 ng/ml) for two hours, and luciferaseactivity in the cells was measured (FIG. 4 depicts a graph showingrelative inhibition activities of compound 1.1 (SEQ ID NO: 2) andcompound 1.2 to NF-κB activation). When the cells were treated withcompound 1.1 (SEQ ID NO: 2) and compound 1.2 according to the presentinvention, activation of NF-κB, which was induced by LPS, was inhibited(FIG. 4).

In addition, 5x NF-κB-Luc reporter plasmid was transfected into RAW264.7 macrophage cells and treated with compounds of the presentinvention, as described above. After 24 hours of the transfection, thecells were pretreated with DMSO (control), compound 1.1 (SEQ ID NO: 2),reference compound 1 (SEQ ID NO: 18) (compound where palmitic acid isremoved from compound 1.1 (SEQ ID NO: 2)), and smaducin-6 were treatedat various concentrations of 100 pM, 1 nM, and 100 nM for 30 minutes,and then the cells were treated with 100 ng/mL of LPS for two hours.Luciferase activity in the cells was measured and the results arepresented in FIG. 5.

As shown in FIG. 5, when the cells were pretreated with compound 1.1(SEQ ID NO: 2) (similar to the cells pretreated with smaducin-6),inhibition of the activity of NF-κB, which was induced by LPS treatment,was increased as dose of compound 1.1 (SEQ ID NO: 2) increased. Incontrast, when the cells were pre-treated with DMSO or referencecompound 1 (SEQ ID NO: 18), the activity of the NF-κB was not inhibitedin dose dependent manner.

Example 2.3 Signaling Pathway Selectivity

To evaluate signaling pathway selectivity of compound 1.1 (SEQ ID NO: 2)according to the present invention, the following experiments wereperformed. The following experiments were performed to test whethercompound 1.1 (SEQ ID NO: 2) specifically suppressed a signaling pathwayinduced by an individual inducer. Specifically, 5×NF-κB-Luc reporterplasmid, SBE-Luc reporter plasmid, and BRE-Luc reporter plasmid wereindividually transfected into Raw 264.7 macrophage cells. After 24hours, the cells transfected with the NF-κB-Luc reporter plasmid weretreated with 100 ng/mL of LPS, the cells transfected with the SBE-Lucreporter plasmid were treated with 5 ng/mL of TFG-β1, and the cellstransfected with the BRE-Luc reporter plasmid were treated with 100ng/mL of BMP6 for two hours.

FIGS. 6A-6C is a graph showing the relative suppression activities ofcompound 1.1 (SEQ ID NO: 2) to different signaling pathways. As shown inFIGS. 6A-6C, when the cells were treated with compound 1.1 (SEQ ID NO:2) according to the present invention, activation of NF-κB, which wasinduced by LPS treatment, was inhibited. However, activation of BRE,which was induced by the BMP6 treatment, or activation of SBE, which wasinduced by the TFG-β1 treatment, was not inhibited by compound 1.1 (SEQID NO: 2). Accordingly, compound 1.1 (SEQ ID NO: 2) selectivelyinhibited activation of NF-κB signaling pathway.

Particularly, recent studies have reported that, for treating aninflammatory bowel disease, TFG-β and BMP signaling pathways mayspecifically relate to mucosal wound healing and the like (Nature 449(2007), 361-365, Am J Path, 162(2), (2003), Nature Immunol. 6, (2005),507-514, J Cell Physiol. 196(2): (2003); 258-64), and Nature Protocols,8(3), (2013) 627-637, which are incorporated herein by reference), andTFG-13 could be a very important factor in control of the inflammatorycondition (dendritic cell conditioning) in intestines (J. Clin. Invest.,111 (2003), 1297-1308, Immunity, 10 (1999), 39-49, Eur. J. Immunol, 36(2006), 864-874, Immunity, 25 (2006), 319-329, Cell 118 (2004), 229-241,and J. Immunol. 179 (2007), 2690-2694, which are incorporated herein byreference). Accordingly, the compounds of the present invention do notsuppress activation of TFG-β3 and BMP signaling pathways which indicatedthat those compounds are markedly effective for the treatment ofinflammatory bowel disease.

Example 2.4 Disruption of Formation of Signaling Complex and

Degradation of Inhibitor κB (IκB)

In order to test whether the compound of the present invention candisrupt formation of inflammation signaling pathway protein complexmediated by MyD88 and/or RIP1, e.g. Toll-like receptors (TLRs) signalingpathway protein complex, immunoprecipitation experiments were performed.At a same time, the following experiments were performed to measure IκBdegradation by the compound of the present invention as measuringchanges of concentration of IκB.

Specifically, using antibodies corresponding to proteins which relate tothe formation of Toll-like receptors (TLRs) signaling pathway proteincomplex (for example, IRAK1, TRAF6, MyD88, RIP1 and Pellino-1),disruption of the formation of inflammatory signaling pathway proteincomplexes was affirmed by immunoprecipitation. As reference control, arelative amount of expression of β-actin from the total cell lysate wascompared and presented using Western blot.

RAW264.7 macrophage cells were individually retreated with compound 1.1(SEQ ID NO: 2) and compound 1.2, and smaducin-6, and further treatedwith LPS. These RAW264.7 macrophage cells were collected, lysed in alysis butter (PBS containing 0.5% Triton X-100, 20 mM HEPES (pH 7.4),150 mM NaCl, 12.5mM β-glycerol phosphate, 1.5 mM MgCl₂, 10 mM NaF, 2 mMDTT, 1 mM Na3O4V, 2 mM EGTA, and 1 mM Protease Inhibitor(PMSF)), andcentrifuged at 13000 rpm for 10 minutes. For immunoprecipitation assays,the supernatant was incubated at 4° C. for 12 hours, with protein-Aagarose beads and the antibodies corresponding to the above proteins,and the beads were subsequently washed three times with the lysisbuffer. The immunoprecipitated substances were dissociated from thebeads with addition of 2x sample buffer, and were boiled. The preparedsamples were loaded on SDS-polyacrylamide gel (FIG. 7).

To measure changes in concentration of IκB, RAW264.7 macrophage cellswere individually pre-treated with compound 1.1 (SEQ ID NO: 2), compound1.2, and smaducin-6 at a concentration of 100 nM, and then treated withLPS. The extracted cell lysate was used for immunoblotting with IκBαantibody, and β-actin was used as reference (FIG. 7).

FIG. 7 shows immunoprecipitation images indicating that compound 1.1(SEQ ID NO: 2) and compound 1.2 disrupted the inflammatory signalingpathway protein complexes, which are mediated by MyD88 or RIP1 andadditionally indicating changes in the concentration of IκB by thesecompounds. When the cells were treated with the compounds according tothe present invention, as compared to the control of total cell lysate,the formation of inflammatory signaling pathway protein complex (whichis mediated by MyD88 or RIP1) was disrupted (FIG. 7). Cells treated withthe compound of the present invention were stabilized bydephosphorylating IκB as compared to expression of the reference ofβ-actin.

When the cells were treated with the compounds according to the presentinvention, as compared to the control of total cell lysate, formation ofthe MyD88 protein complex and the RIP1 protein complex, and activitiesthereof were substantially disrupted and inhibited (FIG. 7). Theseresults indicated that the compounds in the present invention may beused for the treatment of diseases related to Pellino-1. Furthermore, inaddition to above described inflammatory bowel diseases, the compoundsof the present invention may be effective for preventing or treatinggeographic atrophy, wet-age-related macular disease (wet-AMD),dry-age-related macular disease (dry-AMD), diabetic retinopathy,multiple sclerosis (MS), lung inflammation, bacterial pneumonia, viralpneumonia, Diffuse large B-cell lymphoma (DLBCL, GCB type or ABC type),and alopecia (Journal of Clinical Investigation, 124(11), (2014),4976-4988, J Virology, 86(12), (2012), 6595-6604, Nature Medicine,19(5), (2013), 595-602, J. Immunol., 187 (2011), 1-14, J. Inv. Derm.,132 (2012), 43-49, Med. Inflamm., (2010), Article ID 928030, Hair TheTransplant, 4 (2014), 4:1, Exp. Derm., 17 (2007), 12-19, and DDT Dis.Mech. 5 (2009), e163-171), which are incorporated herein by reference).

Bone-marrow-derived macrophage (BMDM) cells were individually pretreatedwith compound 1.1 (SEQ ID NO: 2), reference compound 1 (SEQ ID NO: 18),and smaducin-6 at various concentrations of 100 pM, 1 nM, and 100 nM,and then further treated with 100 ng/mL of LPS. The extracted celllysates were used for immunoblot as described above, and the resultswere shown in FIG. 8.

In case of cells pre-treated with compound 1.1 (SEQ ID NO: 2) ascompared to cells pretreated with smaducin-6, formation of theinflammatory signaling pathway complex (for example, Toll-like receptors(TLR)), which was mediated by MyD88 and RIP1, was further disrupted inan increasing dose dependent manner of compound 1.1 (SEQ ID NO: 2) (FIG.8). Meanwhile, cells pretreated with DMSO or reference compound 1 (SEQID NO: 18) formed the inflammatory signaling pathway complex, which ismediated by MyD88 and RIP1 in a non-dose dependent manner.

Likewise, as compared to the control of total cell lysate, when thecells were pre-treated with compound 1.1 (SEQ ID NO: 2) of the presentinvention, formation of the inflammatory signaling pathway complex,which is mediated by MyD88 and RIP1, was substantially disrupted. Theexperiments using BMDM cells with the compound of the present inventionindicated that the compound of the present invention is effective forpreventing and treating multi diseases relating to MyD88, preventing andtreating diseases relating to expression of Pellino-1 such as viralinfection (respiratory viral infection, viral pneumonia), bacterialpneumonia, autoimmune disease, blood cancer including lymphoma, tumorsin various internal organs (e.g., liver, lung, intestine, prostate,pancreas and the like), and preventing and treating multiple sclerosis(MS).

As depicted in FIGS. 9A and 9B, RAW 264.7 macrophage cells (top FIG. 9A)and BMDM cells (FIG. 9B) were individually pretreated with compound 1.1(SEQ ID NO: 2), reference compound 1 (SEQ ID NO: 18), and smaducine-6 atvarious concentrations of 100 pM, 1 nM, and 100 nM and then furthertreated with 100 ng/ml of LPS. The results were compared with expressionof reference of β-actin. The expression of IκB was increased in thecells as the dose of pretreated compound 1.1 (SEQ ID NO: 2) andsmaducin-6 increased. Meanwhile, IκB was degraded in cells pretreatedwith DMSO or reference compound 1 (SEQ ID NO: 18) because it was shownto be phosphorylated. Accordingly, compound 1.1 (SEQ ID NO: 2) of thepresent invention inhibited the degradation of IκB.

Example 2.5 Evaluating Correlation Between Dose and Disease ActivityIndex

To evaluate the disease activity index of the compounds of the presentinvention in an animal model with chronic colitis induced by dextransulfate sodium (DSS), the following experiments were performed.

Mice (7-8 weeks, female, C57BL/6) were fed 2% DSS polymer (MW of about50000 Da) in drinking water for 5-7 days, and colitis was induced every2 to 15 days. Compound 1.1 (SEQ ID NO: 2) was then administered orallyto the mice, which had induced chronic colitis by DSS, in an amount of50 mg/kg, 100 mg/kg, 200 mg/kg and 400 mg/kg, respectively, from thethird day after feeding DDS, daily, for 11 days. Body weights, diarrheaand hemafecia of the mice were checked daily and the disease activityindexes were measured (FIG. 10).

FIG. 10 is a graph indicating disease activity index scores in theanimal model with DSS-induced chronic colitis according to the dose oforally administered compound 1.1 (SEQ ID NO: 2) and compound 1.2. Asshown in FIG. 10, when compound 1.1 (SEQ ID NO: 2) of the presentinvention was administered at different doses from 50 mg/kg to 400mg/kg, the disease activity thereof was increased as the dose increased,and the disease activity was saturated at a dose of 200 mg/kg.Accordingly, the compound of the present inventionproportionally-increased activity in a dose dependent manner.

Example 2.6 Suppression of Activity of Bowel Disease

To evaluate the supersession activity of the compounds of the presentinvention in an animal model with induced acute colitis (which isinduced by DSS), the following experiments were performed.

Mice (7-8 weeks, female, C57BL/6) were fed 2% DSS polymer (MW of about50000Da) in drinking water for 7-8 days, and colitis was induced. Then,each mouse having acute colitis was administered sulfasalazine in anamount of 500 mg/kg and compound 1.1 (SEQ ID NO: 2) in an amount of 100mg/kg daily for 14 days. Body weights, diarrhea and hemafecia of themice were checked daily and disease activity indexes (DAI) were measured(FIG. 11A).

The DAI was measured as follows:

1) weight loss (0 point: no weight loss; 1 point: reduced weight by1-5%, 2 points: reduced weight by 6-10%, 3 points: reduced weight by11-20% ; 4 points: reduced weight by 20% or greater);

2) diarrhea (0 point: normal bowel movement; 2 points: loose bowelmovement; 4 points: diarrhea); and

3) hemafecia (0 point: normal stool; 2 points: mild blood in stool; 4points: heavy blood in stool).

FIG. 11A is a graph indicating disease activity index scores presentingsuppression activities of the administered compounds in the animal modelwith DSS-induced acute colitis. As compared to anti-inflammatorysulfasalazine, the compound of the present invention had sufficientdisease activity index score at reduced dose, and thus, the compound ofthe present invention is more effective for treating colitis (FIG. 11A).

Meanwhile, as shown in FIGS. 11B-11D, mice suffering from chroniccolitis induced by DSS were orally administered sulfasalazine in anamount of 500 mg/kg and compound 1.1 (SEQ ID NO: 2) in an amount of 100mg/kg daily for 10 days. After 10 days of administration, largeintestine tissues were obtained from the mice, and expression ofchemokines (CCL20, CCL2 and CX3CL1) in the tissues was measured usingreal-time polymerase chain reaction (Real-Time PCR) as described above.Compound 1.1 (SEQ ID NO: 2) was an effective chemokine blockers byinhibiting chemotaxis of pathogenic immune cells into the inflamedtissues.

Example 2.7 Histological Analyses of Colon Villi

To confirm treatment effect of DDS-induced chronic colitis by compound1.1 (SEQ ID NO: 2) of the present invention, large intestinal villi froma non-treated group, a DDS-induced chronic colitis model group and agroup treated with compound 1.1 (SEQ ID NO: 2) were photographed (FIGS.12-16).

FIGS. 12-14 are images showing shapes of the large intestinal villi fromthe non-treated group, the DDS-induced chronic colitis model group andthe group treated with compound 1.1 (SEQ ID NO: 2). As shown in FIGS.12-14, in comparison to the large intestinal villi from the DSS-inducedchronic colitis model, the villi from the group treated with compound1.1 (SEQ ID NO: 2) were similar to the villi of the non-treated group.

FIG. 15 shows photographic images of large intestinal tissues obtainedfrom non-treated group, the DDS-induced chronic colitis model group, thegroup treated with compound 1.1 (SEQ ID NO: 2) (100 mpk) and the grouptreated with sulfasalazine (500 mpk) as an anti-inflammatory drug forcolitis treatment. FIG. 16 shows photographic images of morphology oflarge intestinal mucous membranes obtained from the non-treated group,the DDS-induced chronic colitis model group, the treated group withcompound 1.1 (SEQ ID NO: 2) (100 mpk) and the treated group withsulfasalazine (500 mpk) as an anti-inflammatory drug for colitistreatment, and the mucous membranes were stained with Alcian blue. Asshown in FIGS. 15-16, when the DSS-induced chronic colitis model wastreated with compound 1.1 (SEQ ID NO: 2), histological damages, whichcan be found in inflamed tissues, were alleviated. In addition, incomparison to the non-treated group and the group treated withsulfasalazine, the group treated with compound 1.1 (SEQ ID NO: 2) hadgreater recovery of the mucous membranes by blocking inflamed cells intothe tissue.

FIG. 17 is a graph showing recovery level of large intestinal wall inthe non-treated group, the DDS-induced chronic colitis model group, thetreated group with compound 1.1 (SEQ ID NO: 2) (100 mpk) and the treatedgroup with sulfasalazine (500 mpk).

Specifically, as described in Nature Protocols, 8(3), (2013) 627-637, onthe 8th day 8 of treatment in DDS-induced chronic colitis models,FITC-Dextran was orally administered at dose of 44 mg per 100 g bodyweight. After four hours of administration, blood in an amount of300-400 μL was collected from heart of the mice. The FITC-Dextranreleased in blood stream was measured using a spectrophotofluorometer.

FIG. 17 quantitatively shows that, by analyzing the FITC-Dextranreleased in blood stream, tight junction in large intestinal epithelialtissue caused by embolization of large intestinal mucous membrane, orfunction at the large intestinal wall were recovered as much as thecurrent sulfasalazine treatment. Accordingly, the compound of thepresent invention had a significant effect on recovering the intestinalepithelial barrier and tight junction functions in chronic colitistissues.

Example 2.8 Plasma Concentration

To evaluate changes of the blood (blood stream) concentration ofintravenous and oral administration of compound 1.1 (SEQ ID NO: 2) ofthe present invention, the following experiments were performed.

Compound 1.1 (SEQ ID NO: 2) was administered to a rat intravenously(I.V., 5 mg/kg) or orally (50 mg/kg), and 24 hours after administration,the concentration of compound 1.1 (SEQ ID NO: 2) in plasma was measured(FIG. 18 and FIG. 19). Changes in the concentration of the compound 1.1(SEQ ID NO: 2) when intravenously administered at various time intervalswere measured (FIG. 18). The concentration was decreased to 1/100 of theinitial concentration thereof within 1-2 hours and within 8 hours fromthe administration. Compound 1.1 (SEQ ID NO: 2) was detected in blood atvery low concentration.

Changes in the blood concentration of compound 1.1 (SEQ ID NO: 2) atvarious time courses when orally administered were measured (FIG. 19).After 30 minutes from the oral administration, compound 1.1 (SEQ ID NO:2) was not detected in blood. Table 2 shows pharmacokinetic parametersof compound 1.1 (SEQ ID NO: 2).

TABLE 2 Pharmacokinetic parameters of compound 1.1 (SEQ ID NO: 2)Profile 5 mg/kg (i.v.) 50 mg/kg (p.o.) In vivo Animal Male SD rat MaleSD rat PK Cmax (ng/mL) 6,017.3 ± 2,302.4 0 (Rat, single Tmax (hr) 0.08 0dose) AUClast 2,135.6 ± 848.1  0 (ng · hr/mL) T½ (hr) — 0 Vss (L/kg) 5.4± 1.0 — CL (L/hr/kg) 2.7 ± 1.3 —

Example 2.9 Tissue Distribution

To evaluate tissue distribution of compound 1.1 (SEQ ID NO: 2) accordingto the present invention, the following experiments were performed.

Compound 1.1 (SEQ ID NO: 2) was orally administered to a rat at dose of10 mg/kg, and after 2 hours and 8 hours, concentrations of compound 1.1(SEQ ID NO: 2) were measured in the small intestinal tissue, largeintestinal tissue, appendix tissue, retrimentum in small intestine andretrimentum in large intestine. The results are shown in the followingTable 3.

TABLE 3 Concentration of compound 1.1 (SEQ ID NO: 2) after oraladministration Concentration (ng/mL) Species 2 hours 8 hours smallintestinal tissue  75 ± 70.2 78.1 ± 59.9 large intestinal tissue 30 ± 1033.2 ± 28.8 appendix tissue 22.7 ± 2.56 48.2 ± 46  retrimentum in small1118 ± 217  876 ± 900 intestine retrimentum in large ND 1278 ± 52.7 intestine ND: Not Determined

Quantitation range: intestine 8-2000 ng/kg, retrimentum 30-1000 ng/mL

As shown in Table 3, compound 1.1 (SEQ ID NO: 2) was distributed inintestinal tissues such as small intestinal, large intestinal andappendix tissues at 2 hours and 8 hours from administration in aquantitative range, and further compound 1.1 (SEQ ID NO: 2) wasdistributed in internal tissues 8 hours after administration.

Accordingly, when orally administered, an effective concentration of thecompound of the present invention is continuously maintained inintestinal tissues, even after 8 hours. Because the compound of thepresent invention is a small molecule drug, the compound can be readilytaken up into the intestines such that effective concentration thereofmay be readily reached. Accordingly, by oral administration, thecompound described herein may be used for efficiently treatinginflammatory bowel diseases.

Example 2.10 Inhibition of Activity of MAPK Signaling Pathway

To test whether compound 1.1 (SEQ ID NO: 2) and compound 1.2 of thepresent invention suppress MAPK/ERK signaling pathway, Western blotanalysis was performed (FIG. 20A).

RAW 246.7 macrophage cells were pretreated with compound 1.1 (SEQ ID NO:2), compound 1.2 and reference compound 1 (SEQ ID NO: 18) at aconcentration of 100 nM for 30 minutes and then further treated with LPSfor 0 hour, 0.5 hour, 1 hour and 2 hours. Phosphorylation of MAPKsignaling pathway proteins (ERK1/2, JNK, p38) from the cell treated withcompound 1.1 (SEQ ID NO: 2), and compound 1.2 were activated at 0.5 hourwith the same pattern as the case of reference compound 1 (SEQ ID NO:18), and after 1 hour or 2 hours of treatment, phosphorylation of thoseproteins were gradually decreased with the same pattern as the case withreference compound 1 (SEQ ID NO: 18). β-actin was used as referencecontrol. Changes of the concentration upon phosphorylation of eachprotein were measured by immunoblotting using anti-phosphorylationantibodies. Reference compound 1 (SEQ ID NO: 18), compound 1.1 (SEQ IDNO: 2) and compound 1.2 did not inhibit phosphorylation of the MAPKsignaling pathway proteins.

FIG. 21 shows images determining the inhibition of phosphorylation ofMAPK signaling pathway proteins (ERK1/2, JNK, p38). BMDM cells werepretreated with DMSO, reference compound 1 (SEQ ID NO: 18), compound 1.1(SEQ ID NO: 2) and smaducin-6 at various concentrations of 100 pM, 1 nMand 100 nM and then further treated with LPS 2 hours. Thereafter,Western blot analysis was performed. Compound 1.1 (SEQ ID NO: 2)according to the present invention was not related to suppressingphosphorylation of MAPK/ERK signaling pathway proteins despite increaseof dose, which was similar to DMSO and reference compound 1 (SEQ ID NO:18).

Example 2.11 Comparison with IRAK1/4-Inhibitor

As described in Example 2.3, 5x NF-κB-Luc reporter plasmid wastransfected into RAW 246.7 macrophage cells. After 24 hours, the cellstransfected with NF-κB-Luc reporter plasmid were pretreated withcompound 1.1 (SEQ ID NO: 2) (100 nM),interleukin-1-receptor-associated-kinase-1/4 inhibitor (IRAK1/4)inhibitor (25 μM, CAS 509093-47-4), and smaducin-6 (100 nM) for 30minutes, and then further treated with LPS (100 ng/ml) for 2 hours.Subsequently, luciferase activities in the cells were measured.

FIGS. 22A and 22B show inhibition of NF-κB activation by compound 1.1(SEQ ID NO: 2) (FIG. 22A) and IRAK1/4 inhibitor (FIG. 22B) relatively.At a high IRAK1/4 inhibitor concentration (e.g. 25 μM), NF-κB activationwas inhibited, which was similar to compound 1.1 (SEQ ID NO: 2).

Changes in IκB concentration in the cell lysate of RAW 246.7 macrophagecells, which were pretreated with compound 1.1 (SEQ ID NO: 2) (100 nM),IRAK1/4 inhibitor (25 μM) and smaducin-6 (100 nM) and then furthertreated with LPS (100 ng/ml), were measured (FIG. 23A). RAK1/4 inhibitoralso suppressed degradation of IκB similar to compound 1.1 (SEQ ID NO:2) (FIG. 23B).

FIGS. 24A and 24B shows a graph and images comparing suppression ofMAPK/ERK signaling pathway by compound 1.1 (SEQ ID NO: 2) and IRAK1/4inhibitor. Specifically, RAW 246.7 macrophage cells were pretreated withDMSO, compound 1.1 (SEQ ID NO: 2) (100 nM), IRAK1/4 inhibitor (25 μM),and smaducin-6 (100 nM) for 30 minutes, and then further treated withLPS (100 ng/ml) for two hours. FIG. 24A specifically showsimmunoblotting results to evaluate whether phosphorylation of MAPKsignaling pathway proteins such as ERK, JNK, and p38 were inhibited bythe above treatment. β-actin was used as reference. Compound 1.1 (SEQ IDNO: 2) and smaducin-6 neither suppressed MAPK signaling pathway norinhibited phosphorylation of the proteins. In contrast, IRAK1/4inhibitor suppressed at least one or more of MAPK signaling pathways.

In addition, to check for an unexpected side effect (e.g., from the factthat IRAK1/4 inhibitor suppresses AP-1 transcription signal in MAPKpathway induced with LPS, which is in contrary to compound 1.1 (SEQ IDNO: 2)), the following experiments were performed.

RAW 246.7 macrophage cells were transfected with AP-1-Luc reporterplasmid as described in Example 2.3. After 24 hours, the transfectedcells with AP-1-Luc reporter plasmid were pretreated with compound 1.1(SEQ ID NO: 2) (100 nM), IRAK1/4 inhibitor (25 μM), and smaducin-6 (100nM) for 30 minutes, and then further treated with LPS (100 ng/ml) for 2hours. Luciferase activity in the cells was measured and results arepresented in FIG. 24B. Compound 1.1 (SEQ ID NO: 2) of the presentinvention selectively inhibited activity of NF-κB signaling pathway, butdid not suppress MAPK signaling pathways which is important tobiological activity of cells. However, IRAK1/4 inhibitor inhibited MAPKsignaling pathway, which may result in inhibiting AP-1 transcriptionalfactors, such that, when applied in biological subject, unexpected sideeffects may occur.

The results in Examples 2.1 to 2.11 above indicated that the compoundsof the present invention can (1) inhibit expression of interleukin-6 andactivity of NF-κB, which are induced with LPS treatment; (2) disruptinflammatory signaling pathways mediated by MyD88 and RIP1; and (3)provide similar disease activity index at less dose than the dose ofsulfasalazine that is current anti-inflammatory drug for colitis. Inaddition, when the compounds in the present invention are orallyadministrated, concentration in blood stream is low, whereas theeffective concentration is maintained in cells and/or tissues. Inparticular, in intestinal tissues, the effective concentration thereofcan be maintained even after 8 hours of administration. Accordingly, thecompounds of the present invention are used for treating inflammatorydisease in intestinal tissues, and particularly, are effectively usedfor preventing, alleviating and treating inflammatory bowel disease suchulcerative colitis, Behcet's Disease, Crohn's disease and the like.

Example 2.12 Effect on Retinal Pigment Epithelium Cells

Compound 1.1 (SEQ ID NO: 2) influenced angiogenesis related factors orsuppression factors.

ARPE-19 cells were treated with 5.5mM of glucose as reference control.For experiment group, the ARPE-19 cells were treated with 30 mM ofglucose for 48 hours to induce high blood glucose condition, andsimultaneously treated with DMSO, compound 1.1 (SEQ ID NO: 2) (10 nM),and compound 1.1 (SEQ ID NO: 2) (50 nM). Changes in expression of Nox-4,VEGF, VEGFR1, VEGFR2, Ang1, Ang2, Tie-2, EPO, and EPOR proteins weremeasured by Western blot analysis.

Ang1 and Tie-2, which are factors controlling hemorrhage by reinforcingblood vessel, were increased according to increased concentration ofcompound 1.1 (SEQ ID NO: 2) (FIGS. 25A and 25B). However, expression ofNox4 of producing factor of ROS (reactive oxygen species), VEGF ofinducing factor of new blood vessel, VEGFR1,2, Ang2 of antagonizingfactor for Ang1, and EPO and EPOR of factors for diabetic retinopathywas reduced by treating with compound 1.1 (SEQ ID NO: 2).

In addition, HRMEC cells were treated with 5.5mM of glucose forreference group. For experiment group, the HRMEC cells were treated with30 mM of glucose for 48 hours to induce high blood glucose condition,and simultaneously treated with DMSO, compound 1.1 (SEQ ID NO: 2) (10nM), and compound 1.1 (SEQ ID NO: 2) (50 nM). Changes in expression ofNox-4, VEGF, VEGFR1, VEGFR2, Ang1, Ang2, Tie-2, EPO, and EPOR proteinswere quantitatively measured by qRT-PCR (quantitative RT-PCR).Expression of VEGF precursor as stimulating factor of new blood vesselwas reduced by treatment with compound 1.1 (SEQ ID NO: 2) (FIG. 25C).

To evaluate the effect of compound 1.1 (SEQ ID NO: 2) on tube formationduring angiogenesis, HRMEC cells (8×3) were cultured on Matrigel coatedmicro slides and treated with 20 ng/ml of VEGF for 4 hours to induce thetube formation. Simultaneously, for reference group, the cells weretreated with DMSO, and for experimental group, the cells were treatedwith 50 nM of compound 1.1 (SEQ ID NO: 2) and 1 uM of calcein-AM. Thecells were observed using fluorescence microscope. FIG. 26 shows imagesof tube formation in the cells using fluorescence microscope, which canshow that compound 1.1 (SEQ ID NO: 2) suppressed tube formation. Indeed,in ARPE-19 cells of human retinal pigment epithelium cells, compound 1.1(SEQ ID NO: 2) suppressed expression of Nox-4, VEGF, VEGFR1, VEGFR2,Ang2, EPO, and EPOR proteins according to a concentration gradientthereof, and increased expression of Ang1 and Tie-2 was observed. Assuch, compound 1.1 (SEQ ID NO: 2) is effectively used for preventing,alleviating or treating ophthalmic indications such as diabeticretinopathy. In addition, by disrupting formation of signaling pathwaycomplex of Myd88, the compound 1.1 (SEQ ID NO: 2) is effectively usedfor preventing, alleviating or treating geographic atrophy,wet-age-related macular disease (wet-AMD), dry-age-related maculardisease (dry-AMD) and the like (Cell 149(4), (2012); 847-859).

Example 2.13 Effect on Diabetic Retinopathy

Streptozotocin (STZ) was administered at dose of 50mg/kg to mice dailyfor 5 days, and a mouse model having induced diabetic retinopathy wasobtained. The experimental group of the mouse model was injected withcompound 1.1 (SEQ ID NO: 2), in an amount of 0.2 μg into one eye of themouse, from the 20th day to 24th days from the administration, threetimes with two days of interval. After 50 days of administration, a DRsample that was not treated with compound 1.1 (SEQ ID NO: 2) and a DRsample treated with compound 1.1 (SEQ ID NO: 2) were collected.

The collected retina tissues from each mouse group as described abovewere stained with 5 μM of dihydroethidium and active oxygen rates fromeach group were measured (FIG. 27B). The DR sample injected withcompound 1.1 (SEQ ID NO: 2) showed reduced active oxygen rate ascompared to the non-treated DR sample. Accordingly, compound 1.1 (SEQ IDNO: 2) of the present invention is effectively used for preventing,alleviating or treating ophthalmic indications such as diabeticretinopathy from the biological experiments using mice.

Example 2.14 Effect on Multiple Sclerosis

To confirm the effect of compound 1.1 (SEQ ID NO: 2) on multiplesclerosis, mice (10 weeks old, female) were sensitized with MOG35-55CFUand PTX and an experimental autoimmune encephalomyelitis mouse model wasobtained. Experimental methods were described in Oncotarget, Vol. 7(2016), No. 13, 15382-15393, which is incorporated herein by reference.

As shown in FIG. 28A, compound 1.1 (SEQ ID NO: 2) was hypodermicallyinjected at dose of 1 mg/kg and 40 mg/kg for 25 days once in every otherday. As reference group, 10000 unit of Interferon-beta washypodermically injected for 25 days, once every other day. Clinicalscore were measured and presented. Consequently, compound 1.1 (SEQ IDNO: 2) showed to be effective in the treatment for multiple sclerosis atminimal dose of 1 mg/kg in comparison to the conventional druginterferon-beta currently used for multiple sclerosis treatment.

In addition, when changes in body weights were measured from eachexperiment group, the group treated with compound 1.1 (SEQ ID NO: 2) didnot have reduced weight as compared to the EAE disease model group (FIG.28B). In other words, compound 1.1 (SEQ ID NO: 2), as compared to thecurrent primary drug for treating multiple sclerosis, e.g.interferon-beta, exhibited the same or greater treatment effect andsafety, and thus, is effectively used for preventing, alleviating ortreating multiple sclerosis in experimental autoimmune encephalomyelitismodel. Example 2.15: Effect on septicemia

To confirm the effects of compound 1.1 (SEQ ID NO: 2) on septicemia, 10mice in each group (7-week old, male) were anesthetized, and thereafter,the appendicies were exposed by incision of abdomen. The lower portionof ileocecal valve of the exposed appendix was tied and a single holewas made by using a 22 gage syringe needle. The treated appendix wasreinserted into the abdominal cavity, and grafted using thread to obtaina septicemia-induced mice model (cecal ligation and pucture model, CLPmodel). Experimental methods were described in EMBO Mol Med. (2015) Mar12; 7(5):577-92, which is incorporated herein by reference. Compound 1.1(SEQ ID NO: 2) was hypodermically injected to the treated mice at doseof 1 mg/kg, after 2 hours from the CLP procedure, with 12 hourintervals, three times. As reference control, smaducin-6 washypodermically injected at dose of 12 mg/kg as same method used forcompound 1.1 (SEQ ID NO: 2). Survival rates of the mice from each groupwere measured (FIG. 29). Sham comparative group was not treated afterincision of abdomen and anastomosis, CLP+PBS comparative group wastreated with phosphate buffered saline (PBS) instead of drug compoundafter cecal ligation and puncture model was prepared. As a result,compound 1.1 (SEQ ID NO: 2) (1 mg/kg) was effective for the treatment ofsepticemia, which has not been previously treated with conventionaldrugs at low dosages compared to high dosages of smadudin-6 (e.g. 12mg/kg), with 60% of survival rate. In other words, compound 1.1 (SEQ IDNO: 2) was effective on cecal ligation and puncture (CLP) model,indicating that compound 1.1 (SEQ ID NO: 2) is effectively used forpreventing, alleviating or treating septicemia.

<Formulation 1>Granules

Compound of Formula 1 (SEQ ID NO: 1) 2 g Lactose 1 g

The granules were prepared in accordance with the method known in theart.

<Formulation 2>Tablets

Compound of Formula 1 (SEQ ID NO: 1) 100 mg Corn starch 100 mg Lactose100 mg Stearic magnesium  2 mg

The tablets were prepared in accordance with the method known in theart.

<Formulation 3>Capsules

Compound of Formula 1 (SEQ ID NO: 1) 100 mg Corn starch 100 mg Lactose100 mg Stearic magnesium  2 mg

The capsules were prepared in accordance with the method known in theart. <Formulation 4>Injections

Compound of Formula 1 (SEQ ID NO: 1) 100 mg Mannitol 180 mg Na₂HPO₄•2H₂O26 mg Distilled water 2974 mg

The injections were prepared in accordance with the method known in theart.

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All publications, including but not limited to patents and patentapplications, cited in this specification are herein incorporated byreference as if each individual publication

1. A method for preventing, improving, or treating inflammatory boweldisease, which comprises administering to a subject in need acomposition containing, as an active component, a compound representedby the following Formula 1, an optical isomer thereof, or a saltthereof:

wherein: n is 0, 1, or 2; A is -al-, which is an amino acidindependently selected from the group consisting of alanine, (Ala, A),arginine (Arg, R), asparagine (Asn, N), aspartic acid (Asp, D), cysteine(Cys, C), glutamic acid (Glu, E), glutamine (Gln, Q), glycine (Gly, G),histidine (His, H), isoleucine (Ile, I), leucine (Leu, L), lysine (Lys,K), methionine (Met, M), phenylalanine (Phe, F), proline (Pro, P),serine (Ser, S), threonine (Thr, T), tryptophan (Trp, W), tyrosine (Tyr,Y), and valine (Val, V), both terminal ends of the amino acid beingcoupled to a carbonyl group or an amine group by an amide bond; and R1is a straight chain or branched chain C1-36 alkyl, a straight chain orbranched chain C2-36 alkenyl including at least one double bond, or astraight chain or branched chain C2-36 alkynyl including at least onetriple bond (SEQ ID NO: 1).
 2. The method of claim 1, wherein thecomposition is administered via oral, nasal, pulmonary, rectal, buccal,vaginal, ocular and transdermal routes.
 3. The method of claim 1,wherein the inflammatory bowel disease includes ulcerative colitis,Behcet's disease, and Crohn's disease.
 4. The method of claim 1, whereinthe composition, wherein the composition inhibits formation of aninflammatory signal transduction complex mediated by MyD88, inhibitsformation of an inflammatory signal transduction complex mediated byPellino-1, inhibits formation of an inflammatory signal transductioncomplex mediated by Rip1, suppresses expression of at least one proteinselected from the group consisting of G-CSF, IL-2, SCF, VEGF, CX3CL1,IGFBP5, IGFBP6, IL-1α, IL-1β, IL-6, IL-9, MCP-1, MIP-3α, IL12p40/70,MIG, TNF-α, and VCAM-1, or suppresses activity of N F-κB.
 5. A methodfor preventing, improving, or treating disease or syndrome, whichcomprises administering to a subject in need a composition containing,as an active component, a compound represented by the following Formula1, an optical isomer thereof, or a salt thereof, wherein the disease orsyndrome involves formation of a Pellino-1 induced inflammatory signaltransduction complex containing MyD88, RIP1, or both:

wherein: n is 0, 1, or 2; A is -a1-, which is an amino acidindependently selected from the group consisting of alanine, (Ala, A),arginine (Arg, R), asparagine (Asn, N), aspartic acid (Asp, D), cysteine(Cys, C), glutamic acid (Glu, E), glutamine (Gln, Q), glycine (Gly, G),histidine (His, H), isoleucine (Ile, I), leucine (Leu, L), lysine (Lys,K), methionine (Met, M), phenylalanine (Phe, F), proline (Pro, P),serine (Ser, S), threonine (Thr, T), tryptophan (Trp, W), tyrosine (Tyr,Y), and valine (Val, V), both terminal ends of the amino acid beingcoupled to a carbonyl group or an amine group by an amide bond; and R1is a straight chain or branched chain C1-36 alkyl, a straight chain orbranched chain C2-36 alkenyl including at least one double bond, or astraight chain or branched chain C2-36 alkynyl including at least onetriple bond (SEQ ID NO: 1).
 6. The method of claim 5, wherein thedisease or syndrome is inflammatory bowel disease.
 7. The method ofclaim 6, wherein the inflammatory bowel disease includes ulcerativecolitis, Behcet's disease, and Crohn's disease.
 8. The method of claim5, wherein the disease or syndrome includes multiple sclerosis,psoriasis, sepsis, geographic atrophy, wet age-related macular disease,dry age-related macular disease, diabetic retinopathy, infectious lungdiseases, bacterial pneumonia, viral pneumonia, diffuse large B-celllymphoma, viral infection, autoimmune disease, blood cancer includinglymphoma, and tumors in internal organs.
 9. A method for suppressingexpression of cytokines and/or chemokines, which comprises administeringto a subject in need a composition containing, as an active component, acompound represented by the following Formula 1, an optical isomerthereof, or a salt thereof:

wherein: n is 0, 1, or 2; A is -a1-, which is an amino acidindependently selected from the group consisting of alanine, (Ala, A),arginine (Arg, R), asparagine (Asn, N), aspartic acid (Asp, D), cysteine(Cys, C), glutamic acid (Glu, E), glutamine (Gln, Q), glycine (Gly, G),histidine (His, H), isoleucine (Ile, I), leucine (Leu, L), lysine (Lys,K), methionine (Met, M), phenylalanine (Phe, F), proline (Pro, P),serine (Ser, S), threonine (Thr, T), tryptophan (Trp, W), tyrosine (Tyr,Y), and valine (Val, V), both terminal ends of the amino acid beingcoupled to a carbonyl group or an amine group by an amide bond; and R1is a straight chain or branched chain C1-36 alkyl, a straight chain orbranched chain C2-36 alkenyl including at least one double bond, or astraight chain or branched chain C2-36 alkynyl including at least onetriple bond (SEQ ID NO: 1).